CN105385623A - Actinomycete strain JSX-1 for preventing and curing phytophthora sojae and fungicide thereof - Google Patents
Actinomycete strain JSX-1 for preventing and curing phytophthora sojae and fungicide thereof Download PDFInfo
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Abstract
The invention discloses an actinomycete strain JSX-1 for preventing and curing phytophthora sojae, and belongs to the technical field of plant protection. The strain belongs to streptomycete (Streptomyces sp.) and is named as actinomycete JSX-1. The single colony of the actinomycete strain is circular, white and dry and non-transparent in surface; the hypha is thinner, and grows slowly; the bacterial colony is compact in texture and has a fishy smell. The gram stain of the actinomycete strain is purple, and positive. The JSX-1 fungicide has the controlling effect of 55 to 60 percent on phytophthora sojae. The invention provides the actinomycete strain JSX-1 for preventing and curing phytophthora sojae and the fungicide thereof which are not liable to enable phytophthora sojae to produce resistance, and has no pollution to environment, and the fungicide can be used through root-irrigation after soybean seedling emergence.
Description
Technical field
The present invention relates to a kind of microorganism field, be specifically related to a kind of actinomycetes strain JSX-1 and the microbial inoculum thereof of preventing and treating phytophthora root rot of soybean.
Background technology
Soybean is one of most important crop in agriculture production, and the phytophthora root rot of soybean caused by soyabean phytophthora (Phytophthorasojae) is the worldwide disease of harm Soybean production.This disease is the extremely serious soil-borne disease of a kind of harm, a kind of important external Quarantine Objects of Ye Shi China.Phytophthora root rot of soybean found at Indiana, USA early than 1948, occur in the U.S. and world many countries gradually later, China until 1989 by Shen Chongyao first northeastward soybean producing region be separated to soybean phytophthora, this disease has also occurred in the part soybean producing region of China and has caused serious harm at present.
Soyabean phytophthora can, at soil long-term survival, cause soybean seeds, root and stem rot rotten, all can infect whole breeding time, general underproduction 10-30% soybean, serious generation underproduction 50-70%, the even total crop failure had.If soybean is infected before sprouting, can cause seed decay, after emerging, cotyledon does not open finally withered; If soybean is infected at Adult plant, occur water stain shape foxiness at eustipes part at first, scab is upwards expanded downwards, plant top and the bottom blade turns yellow wilting gradually, and hang contracting in sick portion, scab gradually becomes chocolate, there is water stain shape in root, root system development is bad, causes plant withered death; Soybean is infected at later stages, and its symptom is mainly manifested in lower blade and turns yellow withered, but difficult drop-off, basal part of stem near the ground and stem's scab Vandyke brown, and stem dries up frangibility, easily forms flat pod, flat grain.Soyabean phytophthora has the life history of complexity, Multiple Cycle, and soybean phytophthora, once import into, at lesion long-term surviving, can be difficult to radical cure.
Soyabean phytophthora is under the jurisdiction of oomycetes, although oomycetes habit and nutritional mode similar to fungi, but in fact oomycetes evolution on far different than fungi truly, but it is more similar to primary algae, the pathogenesis of oomycetes also with fungi significant difference, therefore, the normal sterile agent for fungi is invalid to phytophthora.At present, the main prevention and controls of soybean blight is chemical prevention, but because soyabean phytophthora has the features such as variation is complicated, reproduction speed is fast, propagate rapidly, viability is strong, and general chemical prevention pharmacy effect site is single, is easily that this disease develops immunity to drugs; And chemical agent has environmental destruction large, and to the highly toxic defect of people and animals, be therefore badly in need of seeking new prophylactico-therapeutic measures.Develop the emphasis that a kind of non-chemically prevention and controls to environment, person poultry safety has become control phytophthora root rot of soybean, and utilize the actinomycetes in soil and microbial inoculum to be one of the most effective approach to carry out biological control.
At present, a kind of actinomycetes strain and the microbial inoculum thereof that not easily make soybean phytophthora generation resistance, control phytophthora root rot of soybean free from environmental pollution is lacked.
Summary of the invention
Technical problem to be solved by this invention there is provided a kind of the actinomycetes strain JSX-1 and the microbial inoculum thereof that not easily make soyabean phytophthora generation resistance, control phytophthora root rot of soybean free from environmental pollution.
To achieve these goals, the present invention is achieved through the following technical solutions: a kind of actinomycetes strain preventing and treating phytophthora root rot of soybean, and this bacterial strain is streptomycete (Streptomycessp.); Preservation name is called actinomycetes JSX-1.
Further, single bacterium colony of described actinomycetes strain is circular, white, surface dry and opaque; Mycelia is comparatively thin, poor growth, and bacterium colony quality is fine and close, and with mud fishy smell.
Further, described actinomycetes strain gramstaining is purple, is the positive.
The actinomycetes microbial inoculum of control phytophthora root rot of soybean prepared by actinomycetes of the present invention.
Further, the actinomycetes microbial inoculum of described control phytophthora root rot of soybean, its activeconstituents is at least one in following (a), (b) and (c):
Actinomycetes described in (a) claim 1;
Actinomycetic fermented supernatant fluid described in (b) claim 1;
Actinomycetic fermenting culture described in (c) claim 1.
The preparation method of the actinomycetes microbial inoculum of control phytophthora root rot of soybean of the present invention, comprises the steps:
(1) described actinomycetes strain JSX-1 is streptomycete, Actinomycetes species in Gause I substratum on growth, temperature is 28-30 DEG C, and in constant incubator, dark placement is cultivated, and incubation time is 2-4d, obtains actinomycetes strain;
(2) according to described in above-mentioned steps (1), when Actinomycetes species cultivates 2-4d, Actinomycetes species is inoculated in 49-51ml fermention medium, temperature is 28-30 DEG C, rotating speed is shaking culture 70-72h under 160-180r/min, then under rotating speed is 6000-8000r/min, centrifugation 15-20min is carried out, abandon bottom settlings, get fermentation supernatant through the bacterial filter of 0.2 μm, obtain actinomycetes microbial inoculum.
Further, in step (1), described Gause I substratum by Zulkovsky starch 19.9-20.1g, sodium-chlor 0.4-0.6g, saltpetre 0.9-1.1g, dipotassium hydrogen phosphate 0.4-0.6g, magnesium sulfate 0.4-0.6g, ferrous sulfate 0.009-0.011g, agar 15-20g forms, pH is 7.1-7.3, with distilled water, said components is settled to 1000ml, sterilizing 19-21min at temperature is 120-121 DEG C; In step (2), described actinomycete fermentation substratum is by Semen Maydis powder 9.9-10.1g, sucrose 9.9-10.1g, sodium-chlor 1.9-2.1g, ammonium sulfate 2.9-3.1g, calcium carbonate 1.9-2.1g form, and pH is 7.2-7.4, with distilled water, said components is settled to 1000ml, sterilizing 19-21min at temperature is 120-121 DEG C.
Actinomycetes microbial inoculum of the present invention is in the application of control phytophthora root rot of soybean.
Beneficial effect: the invention provides a kind of the actinomycetes strain JSX-1 and the microbial inoculum thereof that not easily make soyabean phytophthora generation resistance, control phytophthora root rot of soybean free from environmental pollution.Actinomycetes microbial inoculum belongs to biotechnological formulation, different from problems such as the chemical resistance that chemical agent causes, is a kind of biological approach of control soybean disease of safety non-pollution, is thus conducive to the No-harmful apple orchard of soybean, meet the requirement of agricultural sustainable development.Peasant or can need not reduce the consumption of the agricultural chemicals of other control soybean phytophthoras in Soybean production process, and then minimizing peasant reduces expenses.Meanwhile, this actinomycetes microbial inoculum can reach 50-60% to the prevention effect of phytophthora root rot of soybean.
Embodiment
Further specific descriptions will be done by specific embodiment to the present invention below, but can not be interpreted as it is limiting the scope of the present invention.
Embodiment 1
The qualification of the 16SrRNA molecule of bacterial strain
Actinomycetes strain JSX-1 carries out activation culture on Gause I flat board at temperature is 28 DEG C, Gause I substratum: Zulkovsky starch 19.9-20.1g, sodium-chlor 0.4-0.6g, saltpetre 0.9-1.1g, dipotassium hydrogen phosphate 0.4-0.6g, magnesium sulfate 0.4-0.6g, ferrous sulfate 0.009-0.011g, agar 15-20g form, and pH is 7.1-7.3, with distilled water, said components is settled to 1000ml, sterilizing 19-21min at temperature is 120-121 DEG C.
Incubation time is observe after 2-4d to find, single bacterium colony of JSX-1 be circular, white, surface dry and opaque; Mycelia is comparatively thin, poor growth, and bacterium colony quality is fine and close, and with mud fishy smell.Bacterial strain JSX-1 gramstaining is purple, is the positive.
It is the most similar that morphological specificity and the common bacteria system identification handbook Streptomyces of bacterial strain JSX-1 belong to actinomycetes (Streptomycessp.).
With JSX-1 genome DNA for template, pcr amplification 16SrRNA obtains the amplified production that length is about 1.5Kb, and this and 16SrRNA most high conservative and consistent length in all bacteriums are that the report of about 1.5Kb coincide.The total length that arrangement sequencing result can obtain the 16SrRNA sequence of carrying out Phylogenetic Analysis is 1485bp, by sequencing result Blast search in the GenBank database of NCBI website, carry out nucleotide homology with listed 16SrRNA sequence to compare, find that 16SrRNA sequence and streptomyces (Streptomycessp.) similarity of bacterial strain JSX-1 are higher, be greater than 99%, in conjunction with the cultural characters result of this bacterial strain, actinomycetes strain JSX-1 is initially identified as streptomyces actinomycetes (Streptomycessp.).
The invention provides a kind of the actinomycetes strain JSX-1 and the microbial inoculum thereof that not easily make soyabean phytophthora generation resistance, control phytophthora root rot of soybean free from environmental pollution.Actinomycetes microbial inoculum belongs to biotechnological formulation, different from problems such as the chemical resistance that chemical agent causes, is a kind of biological approach of control phytophthora root rot of soybean of safety non-pollution, is thus conducive to the No-harmful apple orchard of soybean, meet the requirement of agricultural sustainable development.Peasant or can need not reduce the consumption of the agricultural chemicals of other control soybean phytophthoras in Soybean production process, and then minimizing peasant reduces expenses.Meanwhile, this actinomycetes microbial inoculum can reach more than 60% to the prevention effect of phytophthora root rot of soybean.
Embodiment 2
Soybean phytophthora zoospore suspension preparation method step is as follows:
(1) in V8 vegetables juice, CaCO3 is added according to the ratio of 1: 100 (w/v), then under rotating speed is 6000-8000r/min, centrifugation 8-10min is carried out, abandon bottom settlings, the distilled water of 9 times of volumes is added in centrifuged supernatant, be mixed with the V8 vegetable juice culture of 10%, solid medium adds agar according to the ratio of 1.5-1.8%.Temperature is 120-121 DEG C, and sterilization time is 15-20min.
(2) the soybean phytophthora bacterial strain activated growth on V8 substratum will preserved, temperature is 24-26 DEG C, and in constant incubator, dark placement is cultivated, and the activated growth time is 5-6d.
(3) by the soybean phytophthora bacterial strain of above-mentioned cultivation 5-6d, the mycelia block of 2-3mm × 2-3mm size is cut with scalpel.
(4) above-mentioned for 10-20 block mycelia block is placed in the 10% liquid V8 vegetables juice of 14-16ml, 24-26 DEG C, dark condition is cultivated, and incubation time is 2-3d.
(5) after cultivating 2-3d, carefully provoking soybean phytophthora mycelia with aseptic rifle head, remove the substratum on mycelia, chosen in aseptic water by mycelia and slowly rinse, repeat 3-4 time, is white to mycelia substantially.
(6) above-mentioned mycelia is placed in sterile petri dish, adds 10-15ml aseptic water, uniform fold mycelia.
(7) at temperature is 24-26 DEG C, in constant incubator, dark placement is cultivated, and incubation time is 20-24h, and induction produces zoospore.
(8) zoospore suspension sterilized water is adjusted to concentration and is about 1 × 10
4individual/ml.
A kind of actinomycetes strain JSX-1 preventing and treating phytophthora root rot of soybean of the present invention, this bacterial strain is streptomycete (Streptomycessp.).
Single bacterium colony of described actinomycetes strain is circular, white, surface dry and opaque; Mycelia is comparatively thin, poor growth, and bacterium colony quality is fine and close, and with mud fishy smell.Described actinomycetes strain gramstaining is purple, is the positive.
The actinomycetes microbial inoculum of control phytophthora root rot of soybean prepared by actinomycetes of the present invention.Its activeconstituents is at least one in following (a), (b) and (c):
Actinomycetes described in (a) claim 1;
Actinomycetic fermented supernatant fluid described in (b) claim 1;
Actinomycetic fermenting culture described in (c) claim 1.
The preparation method of the actinomycetes microbial inoculum of control phytophthora root rot of soybean of the present invention, comprises the steps:
(1) be streptomycete by described actinomycetes strain JSX-1, Actinomycetes species in Gause I substratum on growth, temperature is 28 DEG C, and in constant incubator, dark placement is cultivated, and incubation time is 2-4d, obtains actinomycetes strain; Described Gause I substratum is by Zulkovsky starch 19.9-20.1g, sodium-chlor 0.4-0.6g, saltpetre 0.9-1.1g, dipotassium hydrogen phosphate 0.4-0.6g, magnesium sulfate 0.4-0.6g, ferrous sulfate 0.009-0.011g, agar 15-20g forms, pH is 7.1-7.3, with distilled water, said components is settled to 1000ml, sterilizing 19-21min at temperature is 120-121 DEG C;
(2) according to described in above-mentioned steps (1), when Actinomycetes species cultivates 2-4d, Actinomycetes species is inoculated in 49-51ml fermention medium, temperature is 28-30 DEG C, rotating speed is shaking culture 70-72h under 160-180r/min, then under rotating speed is 6000-8000r/min, centrifugation 15-20min is carried out, abandon bottom settlings, get fermentation supernatant through the bacterial filter of 0.2 μm, obtain actinomycetes microbial inoculum.Described actinomycete fermentation substratum by Semen Maydis powder 9.9-10.1g, sucrose 9.9-10.1g, sodium-chlor 1.9-2.1g, ammonium sulfate 2.9-3.1g, calcium carbonate 1.9-2.1g form, and pH is 7.2-7.4, with distilled water, said components is settled to 1000ml, sterilizing 19-21min at temperature is 120-121 DEG C.
Actinomycetes microbial inoculum of the present invention is in the application of control phytophthora root rot of soybean.
The dull and stereotyped prevention effect of actinomycetes microbial inoculum measures
It is for subsequent use that soybean phytophthora bacterial strain cultivates 4-5d in 10%V8 substratum; To actinomycetes microbial inoculum and the V8 substratum Homogeneous phase mixing of the scheduled time be cultured to according to the ratio of 1: 10 (v/v), be prepared into the substratum (to add the V8 substratum of the fermention medium supernatant liquor of equal proportion in contrast) of the actinomycetes microbial inoculum containing actinomycetes JSX-1.Soybean phytophthora mycelia outward flange is got with punch tool (d=5mm), soyabean phytophthora bacterium dish is inoculated in above-mentioned substratum, measure the diameter of phytophthora in postvaccinal 3-10d every 24h, statistics actinomycetes microbial inoculum is to the dull and stereotyped antagonistic effect of soyabean phytophthora.The calculation formula of prevention effect effect is: preventive effect (%)=(control group phytophthora diameter-treatment group phytophthora diameter)/control group phytophthora diameter × 100%.
Table 1 is that the actinomycetes microbial inoculum of actinomycetes JSX-1 is to the changing conditions of the dull and stereotyped inhibiting rate of soybean phytophthora with number of days.
Table 1
As table 1 can draw, dull and stereotyped prevention effect measurement result shows that the growth of JSX-1 actinomycetes microbial inoculum to soyabean phytophthora of cultivation 72h has restraining effect to a certain degree.When inoculating 3d, JSX-1 actinomycetes microbial inoculum is the highest to soybean phytophthora mycelia inhibiting rate, and along with the increase of inoculation number of days, it has a declining tendency to the inhibiting rate of soybean phytophthora mycelia, but inhibiting rate still remains on more than 20%.Dull and stereotyped antagonistic experiment shows that the growth of active substance to soybean phytophthora mycelia that these actinomycetes produce when cultivating 72h of the microbial inoculum of actinomycetes JSX-1 has restraining effect to a certain degree.Mean value ± the standard error of numerical value inhibiting rate in table.
Embodiment 3
Excised leaf prevention effect measures
Actinomycetes microbial inoculum is cultured to predetermined time for subsequent use; This microbial inoculum can fill with root use by 2-3d after emerging.Gather healthy leaves of the same size from the neat soybean plant strain of growth, after being about 9-10s with the polysorbas20 soaking soybean blade that concentration is 0.5%, with sterilized water rinsing gently, taking out and dry in Bechtop.In actinomycetes microbial inoculum, 1min is soaked after blade dries up, take out and dry in Bechtop (with actinomycete fermentation medium supernatant immersion treatment blade in contrast), blade connects 4-6 μ l soybean phytophthora zoospore suspension, and each process repeats 6-8 time.At 24-26 DEG C, relative humidity 80-90%, dark condition measures lesion diameter after cultivating 48h, assessment preventive effect.The calculation formula of preventive effect is: preventive effect (%)=(control group lesion diameter-treatment group lesion diameter)/control group lesion diameter × 100%.
Table 2 is that the actinomycetes microbial inoculum of actinomycetes JSX-1 measures the excised leaf prevention effect of soybean phytophthora.
Table 2
As table 2 can draw, soybean excised leaf experimental result shows that the JSX-1 actinomycetes microbial inoculum of cultivation 72h has prevention effect to a certain degree to soybean phytophthora bacterial strain on excised leaf.After 48h, the in vitro preventive effect of JSX-1 actinomycetes microbial inoculum to soybean phytophthora zoospore is 21.21%.Excised leaf experimental result shows that the microbial inoculum of the actinomycetes JSX-1 active substance that these actinomycetes produce when cultivating 72h has restraining effect to a certain degree to soybean phytophthora.Letter representations different in table is significant difference in 0.05 level.
Embodiment 4
Potted plant prevention effect measures
Embodiment 4 is with the difference of embodiment 3: pour into actinomycetes microbial inoculum to the soybean seedling root growing 2 true leaves, and every strain soybean fills with 2.9-3.1ml (pouring into the seedling of isopyknic fermention medium supernatant liquor with root position for contrast).After filling with root 3d, adopt the method for hypocotyl inoculation 0.9-1.1cm place inoculation soybean phytophthora mycelia block below soybean cotyledon, each process inoculation 20-22 strain soybean plant strain.24-26 DEG C of cultivation, after inoculation 4d, calculate plant sickness rate and prevention effect.Each parameter calculation formula is as follows:
Sickness rate (%)=(disease plant number/total plant number) × 100%
Prevention effect (%)=[(control group sickness rate-treatment group sickness rate)/control group sickness rate] × 100%
Table 3 is that the potted plant prevention effect of actinomycetes JSX-1 actinomycetes microbial inoculum to soybean blight measures.
Table 3
As table 3 can draw, detect after inoculation soybean phytophthora mycelia block 4d, potted plant experiment result shows the microbial inoculum root irrigation through actinomycetes JSX-1, and soybean plant strain effectively can resist the infection of soybean blight, and preventive effect reaches 58.70%; Letter representations different in table is significant difference in 0.05 level.
Can prove according to above each embodiment: be a kind of novel agricultural bacteriocide with the medicament of the effective constituent of actinomycetes microbial inoculum of the present invention.This medicament particularly demonstrates remarkable effect to phytophthora root rot of soybean, and environmental pollution is little, the feature that resistance is low.
More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and application claims protection domain is defined by appending claims, specification sheets and equivalent thereof.
Claims (8)
1. prevent and treat an actinomycetes strain for phytophthora root rot of soybean, it is characterized in that: this bacterial strain is streptomyces (Streptomycessp.), and name is called actinomycetes JSX-1.
2. the actinomycetes strain of control phytophthora root rot of soybean according to claim 1, is characterized in that: single bacterium colony of described actinomycetes strain for circular, white, surface dry and opaque; Mycelia is comparatively thin, poor growth, and bacterium colony quality is fine and close, and with mud fishy smell.
3. the actinomycetes strain of control phytophthora root rot of soybean according to claim 2, is characterized in that: described actinomycetes strain gramstaining is purple, is the positive.
4. the actinomycetes microbial inoculum of control phytophthora root rot of soybean prepared of actinomycetes according to claim 1.
5. the actinomycetes microbial inoculum of control phytophthora root rot of soybean according to claim 4, its activeconstituents is at least one in following (a), (b) and (c):
Actinomycetes described in (a) claim 1;
Actinomycetic fermented supernatant fluid described in (b) claim 1;
Actinomycetic fermenting culture described in (c) claim 1.
6. prepare the method for preventing and treating the actinomycetes microbial inoculum of phytophthora root rot of soybean described in claim 1 or 2, it is characterized in that comprising the steps:
(1) described actinomycetes strain JSX-1 is streptomyces, Actinomycetes species in Gause I substratum on growth, temperature is 28-30 DEG C, and in constant incubator, dark placement is cultivated, and incubation time is 2-4d, obtains actinomycetes strain;
(2) according to described in above-mentioned steps (1), when Actinomycetes species cultivates 2-4d, Actinomycetes species is inoculated in 49-51ml fermention medium, temperature is 28-30 DEG C, rotating speed is shaking culture 70-72h under 160-180r/min, then under rotating speed is 6000-8000r/min, centrifugation 15-20min is carried out, abandon bottom settlings, get fermentation supernatant through the bacterial filter of 0.2 μm, obtain actinomycetes microbial inoculum.
7. the preparation method of actinomycetes microbial inoculum according to claim 1, it is characterized in that: in step (1), described Gause I substratum by Zulkovsky starch 19.9-20.1g, sodium-chlor 0.4-0.6g, saltpetre 0.9-1.1g, dipotassium hydrogen phosphate 0.4-0.6g, magnesium sulfate 0.4-0.6g, ferrous sulfate 0.009-0.011g, agar 15-20g forms, pH is 7.1-7.3, with distilled water, said components is settled to 1000ml, sterilizing 19-21min at temperature is 120-121 DEG C; In step (2), described actinomycete fermentation substratum is by Semen Maydis powder 9.9-10.1g, sucrose 9.9-10.1g, sodium-chlor 1.9-2.1g, ammonium sulfate 2.9-3.1g, calcium carbonate 1.9-2.1g form, and pH is 7.2-7.4, with distilled water, said components is settled to 1000ml, sterilizing 19-21min at temperature is 120-121 DEG C.
8. actinomycetes microbial inoculum described in claim 4 ~ 5 is in the application of control phytophthora root rot of soybean.
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| CN110317735A (en) * | 2019-08-15 | 2019-10-11 | 江苏省农业科学院 | Biological and ecological methods to prevent plant disease, pests, and erosion pythium oligadrum and application |
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| CN106396836A (en) * | 2016-07-28 | 2017-02-15 | 黑龙江八农垦大学 | A biological seed coating agent for controlling soybean phytophthora root rot, a preparing method thereof and a using method of the seed coating agent |
| CN106399152A (en) * | 2016-07-28 | 2017-02-15 | 黑龙江八农垦大学 | Streptomyces having inhibitory effect on phytophthorasojae root rot fungi, biocontrol preparation and preparation method of biocontrol preparation |
| CN106399152B (en) * | 2016-07-28 | 2019-05-31 | 黑龙江八一农垦大学 | The inhibited streptomycete of a kind of pair of soybean phytophthora root rot germ, biological prevention and control agent and preparation method thereof |
| CN106396836B (en) * | 2016-07-28 | 2019-07-05 | 黑龙江八一农垦大学 | Soybean phytophthora root rot prevents and treats biological seed coating agent and preparation method thereof, application method |
| CN106434499A (en) * | 2016-12-13 | 2017-02-22 | 石河子大学 | Actinomycete strain KN37 and application thereof |
| CN106434499B (en) * | 2016-12-13 | 2019-06-07 | 石河子大学 | A kind of actinomycetes strain KN37 and its application |
| CN110317735A (en) * | 2019-08-15 | 2019-10-11 | 江苏省农业科学院 | Biological and ecological methods to prevent plant disease, pests, and erosion pythium oligadrum and application |
| CN113475531A (en) * | 2021-06-30 | 2021-10-08 | 海南金雨丰生物工程有限公司 | Actinomycete complex microbial inoculum and preparation method and application thereof |
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Application publication date: 20160309 |