CN101654659B - Method for prolonging retention period of barley blumeria graminis strain - Google Patents
Method for prolonging retention period of barley blumeria graminis strain Download PDFInfo
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Abstract
The invention discloses a method for prolonging a retention period of a barley blumeria graminis strain, belonging to the technical field of preservation methods of crop pathogenic bacteria. The method comprises the following steps: (1) collecting and propagating monosporous strains; (2) cultivating a test tube seedling of an infected barley variety; (3) inoculating and cultivating the barley test tube seedling; (4) preserving and transplanting the barley blumeria graminis strain, and the like. The method can obviously prolong the retention period of the barley blumeria graminis strain from 8-10 days of a conventional method to 60-70 days and can overcome the defects of easy generation of cross infections, loss of specific pathogenic strains, complicated operation, larger manpower and resource consumption, and the like so as to guarantee the normal development of relevant research works of the pathogenic bacteria of barley powdery mildew. The invention can be popularized and applied to a researching and breeding mechanism of barley diseases.
Description
Technical field
The present invention relates to the technical field of crop pathogens store method, relate in particular to a kind of store method of barley powdery mildew bacteria single-ascospore strain.
Background technology
Barley is one of main food crop in the world, and its sown area is only second to wheat, paddy rice and corn, occupies the 4th.China's barley area and gross output occupy the 5th inferior to paddy rice, wheat, corn and grain.That barley can do is feeding, edible, beer with and protective foods.Barley powdery mildew is one of main disease of barley, mostly occurs in the barley producing region of world's humidity, semi-humid, in China most of barley producing region generation is arranged all, mainly occurs in the winter barley district, and is especially heavier in southwest and southeastern coastal areas morbidity.In recent years, because the nitrogenous fertilizer usage quantity increases, planting density increases, the single and warm winter climatic influences of varieties of plant, and the barley powdery mildew evil is serious day by day, can cause 20～30% production loss.
Barley powdery mildew is the fungal disease that is caused by Bu Shi white powder germ barley specialized form (Blumeria graminisf.sp.Hordei L. is called for short Bgh), and its pathogenic bacteria is the live body bacterial parasite of specialization, belongs to the Ascomycetes Erysiphales.This germ main harm blade also can endanger leaf sheath, stem stalk and fringe portion when serious, from seedling to becoming strain, all can be by infection process.The cause of disease mycelia enters with haustorium and absorbs nutrient and moisture growth expansion in the host cell, after the host surface is covered by mycelium, influenced photosynthesis on the one hand, its stratum corneum is by breakage on the other hand, and the effect of pore is lost, respiration and transpiration are out of control, thereby cause the complete stool malnutrition, wheat seeding early ageing, easily lodging, wheat is not plump thin, and output reduces.Exist the relation of " gene pairs gene " between barley powdery mildew bacteria and the host, thereby can utilize the barley differential host of containing different resistant genes that big wheat powdery mildew single-ascospore strain is divided into different pathotypes.But the variation of the toxicity of barley powdery mildew bacteria is very fast, and along with the big area of specialization disease-resistant variety is promoted, its varietal resistance will be lost in several years.Therefore the toxicity structure by analyzing big wheat powdery mildew colony, distribution and dynamically, but the effective area that the enantiopathy gene plays a role estimate and predict, for perspective breeding provides foundation.With separating the different barley powdery mildew bacteria pathotype inoculation barley varieties of preserving, can comprehensively, accurately estimate the disease resistance of kind, identify the mildew-resistance gene of barley variety, seek new resistance source, can effectively control barley powdery mildew by reasonable utilization and layout disease-resistant gene.
Separating and preserve different barley powdery mildew bacteria pathotype bacterial strains, is the prerequisite of carrying out barley powdery mildew bacteria virulent gene genetics research and cause of disease and the research of host's interaction.But barley powdery mildew bacteria is a kind of obligate parasite, can only grow on the host tissue of living.This bacterium can be divided into sexual growth phase and asexual growth phase the life history.Wherein, the barley powdery mildew bacteria nourishing body of asexual growth phase is a mycelium, and is mainly parasitic on the Folium Hordei Vulgaris surface, carries out vegetative propagation by producing conidium, and from the flora in the asexual later stage of growing, produce sexual propagation organ cleistothecium, thereby enter sexual growth phase.After spermary wherein and ascogonium cooperate, produce thecaspore, enter into asexual growth phase again behind the aerial mycelium after thecaspore grows by plasmogamy, caryogamy and reduction division.Under suitable temperature, humidity and illumination condition, the conidium that the asexual growth phase of barley powdery mildew bacteria produces can colonize on the blade of viable specific barley variety, and can repeat to produce; And barley sexual propagation organ cleistothecium produced in the asexual later stage of growing of mycelia, and thecasporous generation then needs by plasmogamy, caryogamy and reduction division, so it raises up seed and can morph, generally should not preserve bacterial strain by sexual propagation.Therefore, the barley powdery mildew bacterial strain mainly is to utilize its conidium to infect susceptible live body barley leaves, and conidial mode of transferring on the live body barley leaves is at set intervals preserved.Because big wheat powdery mildew can constantly absorb nutrition in Mai Yeshang carries out the process of asexual growth, cause tissue necrosis, saprophytic microorganism multiplies procreation, can pollute inactivation in the nourishing body short period of time, is difficult to long-time preservation, can only preserve under the normal condition 8-10 days; Conidium need expend a large amount of human and material resources on the one hand in frequent switching process simultaneously, tends to produce aleopation on the other hand, causes specific pathotype bacterial strain losing unintentionally.In addition, also having about preservation wheat powdery mildew bacterial strain method in the present bibliographical information: (1) on stripped leaf section, places the water agar culture dish that fills preservation agent with inoculation, adds a cover, and can preserve at low temperatures 40 days; (2) with inoculation to test-tube plantlet, can preserve under the low temperature 40 days, but concrete working method is not seen detailed description; The wheat seeding sleeve pipe of (3) growing in the basin alms bowl is isolated, inoculation to the wheat seeding of growth, can be preserved low temperature under 60 days, but this method occupation space greatly, big energy-consuming, need take out the blade that carries disease germs during switching, time-consuming and be easy to generate aleopation.
Therefore, people urgently wish under laboratory condition, seek a kind of easy and simple to handle, time saving and energy saving, energy-conservation, and can reduce aleopation, significantly prolong the method for barley powdery mildew bacterial strain shelf time, so that carry out the research of the variation of big wheat powdery mildew toxicity and host and pathogenic bacteria interaction.
Summary of the invention
The present invention seeks to, short, time-consuming at the former bacterium of existing barley powdery mildew existing preservation period on the barley live body is preserved, take a lot of work and the easy defective of generation aleopation etc. in frequent switching process, propose a kind of easy and simple to handle, time saving and energy saving, and can reduce aleopation between different strains, the method that can the long period preserve the barley powdery mildew bacterial strain is to guarantee normally carrying out of barley powdery mildew pathogenic bacteria correlative study work.
The object of the invention is achieved through the following technical solutions.
A kind of method that prolongs the barley powdery mildew bacteria preservation period, this method is carried out according to the following steps:
(1) collection of single-ascospore strain with expand numerous: the sowing wide spectrum was high earlier in 8-10 days before bacterial strain is gathered feels one heart stage of barley powdery mildew bacteria kind to two leaf, standby; Gather the blade that has single or multiple clear mycelium and still do not have the spore generation from the field, this blade is cut into the leaf section that single hop or multistage respectively have the long 2-3cm of single monospore bacterium colony, and face up by carrying disease germs and respectively to put into a culture dish that fills preservation agent, add a cover, under 18-22 ℃, natural lighting, cultivate 1-2d and produce conidium to the monospore bacterium colony; Other gets the fresh leaf section that high first leaf of feeling barley powdery mildew bacteria kind plant of above-mentioned wide spectrum is cut into long 2-3cm, puts into the culture dish that fills the same preservation agent by one section/every ware; Under Bechtop isolation condition, produced conidial leaf section by a ware/one section with above-mentioned, place the fresh leaf section the top, beat several down after, add a cover; To inoculate the back culture dish and put into illumination box, in 16-18 ℃, relative humidity 90%-95%, every day, 16h expanded numerous cultivation 8-10d under the 2500-3000lx illumination condition, and is to a large amount of generation of conidium are arranged, standby;
(2) cultivation of susceptible barley test-tube plantlet: with the high sense of wide spectrum barley powdery mildew bacteria variety seeds, after the distilled water seed soaking, filter is done, and cultivates 24 hours to germinateing for 18 ℃, and is standby; Other prepares 28 centimetres of a collection of length, 5 centimetres of diameters, and the bottom is provided with 0.5 centimetre of aperture, and the mouth of pipe is made into the test tube of straight mouthful and the turned welt that can mate butt joint respectively, and is standby; Nutrition soil in sterilization back is contained in the identical test tube of orifice configuration, and to 1/4 of every test tube pipe range, nutritive medium irrigates, put into a 8-10 grain germination barley seed/test tube after, cover thin layer nutrition soil again, seal with no mycoderm; Cultivate 8-10 days to 1 heart stage of wheat seeding 2 leaves through 18-22 ℃, standby;
(3) inoculation of barley test-tube plantlet and cultivation: get vertical the placing on the Bechtop of step (2) test-tube plantlet, earlier squirt blade with nutritive medium, clamping step (1) with tweezers again expands and to have a large amount of big conidial leaf sections of wheat powdery mildew after numerous, stretch in the test tube mouth, with after spore inoculating is to the test-tube plantlet, the mouth of pipe is sealed with no mycoderm by vibration; Put into illumination box, in 16-18 ℃, every day 16h, 2500-3000lx illumination cultivation 24h; With 75% alcohol Bechtop is sterilized after the inoculation of a barley test-tube plantlet of every end, in case produce aleopation during next inoculation;
(4) preservation of barley powdery mildew bacterial strain and switching: the test-tube plantlet of step (3) through inoculation, after cultivating placed incubator, at 5 ℃, every day 16h, this bacterial classification can grow, preserve 60-70 days under the 2500-3000lx illumination; After generally being saved to 60 days, the test-tube plantlet opening mouth that is about to this sense bacterium also is inverted, other gets the orifice configuration of a step (2) and the barley test-tube plantlet that this sense bacterium test-tube plantlet mouth of pipe is complementary, the two test tube mouths of pipe connect to place its below also to incite somebody to action up and down, big wheat powdery mildew spore is transferred on the test-tube plantlet of below by vibration, the mouth of pipe is sealed with no mycoderm after sterilizing with 75% alcohol; After the Bechtop sterilization, under same upper type, condition, carry out switching, cultivation and the preservation of other bacterial strain; So circulation is preserved the barley powdery mildew bacterial strain for a long time to realize the laboratory.
Described preservation agent by 1% water agar and benzoglyoxaline in 1000g: the 40mg ratio is formulated.
Described nutritive medium is by 1g/L NaNO
3, 0.25g/L KNO
3, 0.25g/L MgSO
4, 0.25g/L KH
2PO4,0.01g/L FeCl
3, surplus is that distilled water is formulated.
Described nutrition soil is the organic substratum of full element, and slant acidity is produced by peat composed of rotten mosses base, Qinghe, Changbai Mountain, northeast, and through Autoclave, 121 ℃ of heating were used after 30 minutes.
The invention has the beneficial effects as follows:
(1) the test tube mouth of pipe matching structure of the designed proposition of the present invention and the method inoculated with the test-tube plantlet live body, this was both simple and practical, and was easy to operate, had improved inoculation efficient greatly, had effectively prevented the aleopation between different strains again; As in vitro sowing a quantity of seeds, utilize an amount of nutrition soil and nutritive medium to cultivate wheat seeding, wheat seedling growth is healthy and vigorous, and well developed root system is firmly protected soil, though test-tube plantlet needs to be inverted operation during inoculation, native and seedling still can not come off; Test tube bottom is provided with 0.5 centimetre aperture, can get rid of unnecessary nutritive medium or moisture, immerses in the water and can be replenished by this aperture suction at the bottom of only needing pipe when the test-tube plantlet lack of water, and operation is convenient, and is time saving and energy saving, and the minimizing aleopation; The present invention simultaneously will preserve the bacterial strain test-tube plantlet leaves in the illumination box, has to take up room for a short time, and power consumption has obviously reduced the retain costs of bacterial classification less.
(2) cardinal principle of the present invention's utilization is to adopt the test-tube plantlet live body to inoculate on the one hand, and can constantly keep the skin wet with nutrition has postponed the downright bad time of inoculation carrier greatly; Be to inoculate at normal temperatures, cultivate in 24 hours earlier on the other hand, when generating to produce, white powder germ spore germ tube invades silk, after invading wheat leaf epidermal cell formation primary haustorium, in time reduced the storage temperature (5 ℃) of sense bacterium test-tube plantlet, this had both made the slowly growth at low temperatures of wheat seeding and bacterial strain, had also more postponed decaying the time of wheat seeding tissue, thereby had obtained the beneficial effect of obvious prolongation retention period of barley blumeria graminis strain, by 8-10 days of ordinary method, extend to 60-70 of the present invention days.
Embodiment
The present invention is described in further detail by following examples, but content of the present invention is not limited thereto.
Nutrition soil: the complete organic substratum of element, slant acidity, peat composed of rotten mosses base, Qinghe, Changbai Mountain, northeast produces;
Benzoglyoxaline: chemical pure, Chemical Reagent Co., Ltd., Sinopharm Group;
Agar: chemical pure, Beijing ancient cooking vessel state Bioisystech Co., Ltd;
NaNO
3: analytical pure, Hangzhou Fine Chemical Co., Ltd;
KNO
3: analytical pure, Shantou Xilong Chemical Industry Co., Ltd;
MgSO
4: analytical pure, Wenzhou City chemistry materials factory;
KH
2PO
4: analytical pure, lake, Huzhou examination chemical reagent company limited;
FeCl
3: analytical pure, Chemical Reagent Co., Ltd., Sinopharm Group;
No mycoderm: Shanghai is sowed rich garden supplies company limited and is produced;
The high sense of barley wide spectrum Powdery Mildew kind: Zhejiang show 12, Zhejiang Academy of Agricultural Science crop and nuclear technique utilize institute's barley branch center breeding, evaluation.
Embodiment 1:(method 1 that prolongs the barley powdery mildew bacteria preservation period)
This method is carried out according to the following steps:
(1) collection of single-ascospore strain with expand numerous: before bacterial strain is gathered 8-10 days earlier sowing wide spectrum high sense barley powdery mildew bacteria kind " Zhejiang show 12 " to two leaves after date wholeheartedly, standby; Only have single clear mycelium from the field collection, and still do not have the blade that spore produces; From blade, cut the leaf section that has the long 2-3cm of this single monospore bacterium colony, and face up in carrying disease germs to put into and fill by 1% water agar and benzoglyoxaline in 1000g: the culture dish of the formulated preservation agent of 40mg ratio, add a cover, put under room temperature 18-22 ℃, natural lighting condition and cultivate 1-2d, promptly fungal infection takes place and ruins in this leaf Duan Shangwei, and the monospore bacterium colony is when having produced conidium; Other gets first leaf of the high sense of above-mentioned two leaves, one heart stage wide spectrum kind plant, and blade is cut into the fresh leaf section 3-5 section of long 2-3cm, under the isolation condition, puts into the culture dish that fills the same preservation agent respectively by one section/every ware; On Bechtop, the conidial leaf section of generation that above-mentioned field is gathered is by a ware/one section, place the fresh leaf section the top, knock inoculate under several after, add a cover; To inoculate the back culture dish and put into illumination box, in 16-18 ℃, relative humidity 90%-95%, every day, 16h expanded numerous cultivation 8-10d under the 2500-3000lx illumination condition, and to a large amount of generation of conidium are arranged, and that blade is still held is green, not by fungal infection, standby;
(2) cultivation of susceptible barley test-tube plantlet: with the high sense of wide spectrum barley powdery mildew bacteria variety seeds, with (grain weight is determined according to the bacterial strain quantity of switching preservation) behind the distilled water seed soaking 24h, the filter solid carbon dioxide divides the back seed to put into beaker again, seed upper cover two layers of filter paper is put into biochemical incubator, cultivate for 18 ℃ and germinateed in 24 hours, standby; Other prepares 28 centimetres of a collection of length, 5 centimetres of diameters, and the bottom is provided with 0.5 centimetre of aperture, and the mouth of pipe is made into straight mouthful and the turned welt that can mate butt joint respectively, and is standby; The nutrition soil that peat composed of rotten mosses base, Qinghe, Changbai Mountain, northeast is produced is after 121 ℃ of heating of Autoclave sterilization in 30 minutes, contain in straight mouth or the turned welt test tube to 1/4 of pipe range, nutritive medium irrigates to partly flowing out from aperture, after putting into 8-10 grain germination barley seed by every, cover thin layer nutrition soil, the mouth of pipe is sealed with no mycoderm; Cultivated 8-10 days through 18-22 ℃, to 1 heart stage of wheat seeding 2 leaves, standby;
Described nutritive medium is by 1g/L NaNO
3, 0.25g/l KNO
3, 0.25g/l MgSO
4, 0.25g/l KH
2PO4,0.01g/l FeCl
3, surplus is that distilled water is formulated;
(3) inoculation of barley test-tube plantlet and cultivation: get vertical the placing on the Bechtop of step (2) test-tube plantlet, earlier squirt blade with the same nutritive medium, using tweezers (alcohol disinfecting through 75%) to clamp step (1) again expands and to have a large amount of big conidial leaf sections of wheat powdery mildew after numerous, stretch into 5 centimeters in the test tube mouth, by vibrating spore inoculating to test-tube plantlet, the mouth of pipe seals with no mycoderm after 75% wipes of alcohol is crossed sterilization; Put into illumination box,, cultivate 24h under the 2500-3000lx illumination 16h condition, to spore-germination and invade the wheat leaf epidermal cell in 16-18 ℃; With 75% alcohol sprayed in space on the Bechtop after the inoculation of a barley test-tube plantlet of every end, fall the spore sterilization,, prevent aleopation in order to next inoculation;
(4) preservation and the switching of big wheat powdery mildew strain: the carry disease germs test-tube plantlet of step (3) through inoculation, after cultivating put into illumination box, cultivate down, can grow, preserve 60-70 days in 5 ℃, every day 16h, 2500-3000lx illumination; General after being saved to 60 days, be about to feel the test-tube plantlet opening mouthful also inversion of bacterium, other gets the orifice configuration (as straight mouthful) of a step (2) and the barley test-tube plantlet that this sense bacterium test-tube plantlet mouth of pipe (as turned welt) is complementary, the mouth of pipe of two test tubes connects to place its below also to incite somebody to action up and down, big wheat powdery mildew conidium is transferred on the test-tube plantlet of below by vibration, after the mouth of pipe was sterilized with 75% alcohol, the aseptic film that seals sealed; And with 75% alcohol to space spraying on the Bechtop, fall the spore sterilization, preserve in order to next inoculation, prevent aleopation; Preserve after about 60-70 days after the same method is cultivated again, use with the method switching again, preserve, so circulation can be preserved the barley powdery mildew bacterial strain for a long time in the laboratory.
Embodiment 2:(method 2 that prolongs the barley powdery mildew bacteria preservation period)
The present embodiment method, remove in step (1) and gather the blade that has a plurality of clear mycelium and still do not have the spore generation from the field, and this blade is cut into multistage respectively has outside the leaf section of the long 2-3cm of single monospore bacterium colony, remaining step, method, technology are same as embodiment 1.
Claims (4)
1. method that prolongs the barley powdery mildew bacteria preservation period is characterized in that carrying out according to the following steps:
(1) collection of single-ascospore strain with expand numerous: the sowing wide spectrum was high earlier in 8-10 days before bacterial strain is gathered feels one heart stage of barley powdery mildew bacteria kind to two leaf, standby; Gather the blade that has single or multiple clear mycelium and still do not have the spore generation from the field, this blade is cut into the leaf section that single hop or multistage respectively have the long 2-3cm of single monospore bacterium colony, and face up by carrying disease germs and respectively to put into a culture dish that fills preservation agent, add a cover, under 18-22 ℃, natural lighting, cultivate 1-2d and produce conidium to the monospore bacterium colony; Other gets the fresh leaf section that high first leaf of feeling barley powdery mildew bacteria kind plant of above-mentioned wide spectrum is cut into long 2-3cm, puts into the culture dish that fills the same preservation agent by one section/every ware; Under Bechtop isolation condition, produced conidial leaf section by a ware/one section with above-mentioned, place the fresh leaf section the top, beat several down after, add a cover; To inoculate the back culture dish and put into illumination box, in 16-18 ℃, relative humidity 90%-95%, every day, 16h expanded numerous cultivation 8-10d under the 2500-3000lx illumination condition, and is to a large amount of generation of conidium are arranged, standby;
(2) cultivation of susceptible barley test-tube plantlet: with the high sense of wide spectrum barley powdery mildew bacteria variety seeds, after the distilled water seed soaking, filter is done, and cultivates 24 hours to germinateing for 18 ℃, and is standby; Other prepares 28 centimetres of a collection of length, 5 centimetres of diameters, and the bottom is provided with 0.5 centimetre of aperture, and the mouth of pipe is made into the test tube of straight mouthful and the turned welt that can mate butt joint respectively, and is standby; Nutrition soil in sterilization back is contained in the identical test tube of orifice configuration, and to 1/4 of every test tube pipe range, nutritive medium irrigates, put into a 8-10 grain germination barley seed/test tube after, cover thin layer nutrition soil again, seal with no mycoderm; Cultivate 8-10 days to 1 heart stage of wheat seeding 2 leaves through 18-22 ℃, standby;
(3) inoculation of barley test-tube plantlet and cultivation: get vertical the placing on the Bechtop of step (2) test-tube plantlet, earlier squirt blade with nutritive medium, clamping step (1) with tweezers again expands and to have a large amount of big conidial leaf sections of wheat powdery mildew after numerous, stretch in the test tube mouth, with after spore inoculating is to the test-tube plantlet, the mouth of pipe is sealed with no mycoderm by vibration; Put into illumination box, in 16-18 ℃, every day 16h, 2500-3000lx illumination cultivation 24h; With 75% alcohol Bechtop is sterilized after the inoculation of a barley test-tube plantlet of every end, in case produce aleopation during next inoculation;
(4) preservation of barley powdery mildew bacterial strain and switching: the test-tube plantlet of step (3) through inoculation, after cultivating placed incubator, at 5 ℃, every day 16h, this bacterial classification can grow, preserve 60-70 days under the 2500-3000lx illumination; After generally being saved to 60 days, the test-tube plantlet opening mouth that is about to this sense bacterium also is inverted, other gets the orifice configuration of a step (2) and the barley test-tube plantlet that this sense bacterium test-tube plantlet mouth of pipe is complementary, the two test tube mouths of pipe connect to place its below also to incite somebody to action up and down, big wheat powdery mildew spore is transferred on the test-tube plantlet of below by vibration, the mouth of pipe is sealed with no mycoderm after sterilizing with 75% alcohol; After the Bechtop sterilization, under same upper type, condition, carry out switching, cultivation and the preservation of other bacterial strain; So circulation is preserved the barley powdery mildew bacterial strain for a long time to realize the laboratory.
2. by the described method of claim 1, it is characterized in that described preservation agent by 1% water agar and benzoglyoxaline in 1000g: the 40mg ratio is formulated.
3. by the described method of claim 1, it is characterized in that described nutritive medium is by NaNO
3, KNO
3, MgSO
4, KH
2PO4, FeCl
3, distilled water is formulated.
4. by the described method of claim 1, it is characterized in that described nutrition soil is the organic substratum of full element, slant acidity is produced by peat composed of rotten mosses base, Qinghe, Changbai Mountain, northeast, and through Autoclave, 121 ℃ of heating were used after 30 minutes.
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| CN102972302B (en) * | 2012-12-27 | 2013-11-27 | 新疆农业科学院哈密瓜研究中心 | Method for preserving melon powdery mildew fungus |
| CN103004325B (en) * | 2012-12-28 | 2014-06-18 | 青岛啤酒股份有限公司 | Control method for barley safe storage |
| CN103224888B (en) * | 2013-05-13 | 2015-01-07 | 湖北省农业科学院植保土肥研究所 | Long-term preservation method of blumeria graminis strains |
| CN105349432B (en) * | 2015-11-11 | 2019-02-05 | 中国农业大学 | A kind of monospore expanding propagation method of Puccinia polysora Underw |
| CN108641962B (en) * | 2018-03-23 | 2022-02-18 | 湖北省农业科学院植保土肥研究所 | Method for prolonging in-vitro preservation time of wheat powdery mildew |
| CN108977363A (en) * | 2018-07-19 | 2018-12-11 | 河南省农业科学院植物保护研究所 | Test wheat powdery mildew culture preparation method |
| CN109136325B (en) * | 2018-08-22 | 2022-06-03 | 新疆农业科学院植物保护研究所 | Method for providing powdery mildew source for wheat disease resistance identification and wheat powdery mildew disease resistance identification |
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