Background technology: being extensive use of of chemical fertilizer and agricultural chemicals makes a great contribution to agricultural development, is still one of main means that improve crop yield so far.But life-time service chemical pesticide and chemical fertilizer have also brought such as problems such as environmental pollution, ecological damage, sick worm resistance and food safeties, are seriously restricting the Sustainable development of agricultural.Bio-feritlizer, biological pesticide and transgenic plant are major measure of contemporary crop high-quality, efficient, nuisanceless production.Biological pesticide in the past all is that bio-extract or secretory product itself directly play a role, as jinggangmeisu is exactly water soluble antibiotics---the direct killing pathogenic bacteria of glucoside compounds that is produced by the mutation of streptomyces hygroscopicus well hilllock, still welcome on producing so far.Modern age molecular biotechnology fast development, producing biological pesticide and transgenic plant by modes such as gene clone, conversions becomes the focus that people pay close attention to.In recent years the research of BT and PI transgenic plant is succeedd.But the safety issue of transgenic plant is subjected to scientist's great attention, and many countries strictly control the application of this class material.China scientific worker finds that the various plants extract induces potato that the resistance of phytophthora infestans is had good effect, and the disease protection ratio reaches 54%, and can promote that potato seed is sprouted, early growth.The ZhongminWei of Cornell Univ USA finds a kind of protein (Harpin) that causes allergic reaction of plant that plant pathogenetic bacteria Erwinia amylovora produces, molecular weight is 40kd, form by 385 amino acid, the expression of series of genes in the energy inducing plant body, the growing system of activated plant self and defense mechanism, thereby resist the invasion and attack of common disease insect pest and the influence of poor environment, have the root of hair of increasing amount, leaf area, setting percentage simultaneously, effects such as premature ripening and volume increase.U.S. EDEN Biological Science Co., Ltd uses this technological development to go out broad spectrum harmless boilogical agricultural chemicals Messenger, on multiple field crop, cash crop, use, effect is all very obvious, generally can reduce medication 50-80%, volume increase 10-20%, find that now plant pathogenetic bacteria all has the hrp gene cluster, molecular weight 20-40kd comprises 3-13 gene.A lot of cause of diseases of phytophthora (Phophthora) can be secreted and be induced non-host to produce anaphylactoid protein, are called Elictin, and molecular weight is 10.6KD, and it can also bring out various plants simultaneously and produce multiple defense response, improves the disease resistance of these plants.But can other plant pathogenic fungi produce similar protein on host and non-host, do not appear in the newspapers as yet.
Summary of the invention: the present invention is intended to from plant pathogenic fungi the separation and Extraction plant and uses multifunctional protein, the self-defense system and the growing system of inducing plant, thus the invasion and attack of disease and pest resisted, promote growth and development of plants simultaneously, improve crop yield, and promote fruit fresh-keeping.
The present invention is such realization: plant is used multi-functional true mycoprotein goods, be a kind of from plant pathogenic fungi extraction separation, the protein of inducing plant information conduction, it has comprised molecular weight between 42-60KD, active 4 kinds of activated proteins of complementary and molecular weight are the auxiliary activity albumen of 30KD and 20KD, plant pathogenic fungi is cultivated through secondary in conventional nutrient solution, during to the fungal spore maximum, with 5000rpm centrifugal 30 minutes, the spore of collecting is pressed 5mM potassium phosphate buffer or the citrate buffer solution that nutrient solution volume 1/10 adds PH6.8, fungal spore suspension adopts ultrasonic wave 30 minutes or the broken spore cell of 180bar high pressure, centrifugal 30 minutes once more with 5000rpm, remove sedimentary smudge cells tissue, supernatant liquor is promptly made the mycoprotein goods with 100 degree boiling water water-baths 15 minutes.
Be described in further detail the present invention below:
With plant pathogenic fungi Staphlosporonites (Botrytis), interlink spore genus (Alternaria), rice blast fungus (Pyrcularia), poison mould (Penicillium), Aspergillus flavus (Asporgillus), in sheath blight fungus (Rhizoctonia solani) and the Penicillium notatum (Fusarium) any is material, in conventional nutrient solution, cultivate through secondary, during to the fungal spore maximum, through 5000rpm centrifugal 30 minutes, the spore of collecting is pressed 5mM potassium phosphate buffer or the citrate buffer solution that nutrient solution volume 1/10 adds PH6.8, fungal spore suspension adopts ultrasonic wave 30 minutes or the broken spore cell of 180bar high pressure, centrifugal 30 minutes once more with 5000rpm, remove sedimentary smudge cells tissue, keep the mycoprotein in the supernatant liquor, supernatant liquor is spent boiling water water-baths 15 minutes with 100, to kill viable bacteria, make unstable and inactive protein sex change, through the supernatant liquor of water-bath centrifugal 30 minutes again with 5000rpm, the viable bacteria that removal is killed and the albumen of sex change, the supernatant liquor that keeps is mycoprotein, these mycoprotein goods are that molecular weight is 4 kinds of activated proteins between the 42-60KD, and molecular weight is the auxiliary activity albumen of 30KD and 20KD.
Mycoprotein is by the plant surface protein receptor, but the conduction of the information of inducing plant starts a series of metabolic reactions in the plant materials, cause genetic expression, activated plant self-defence system and growing system, thus generation is to the resistance of disease and pest, promote plant-growth, improve crop yield.Mycoprotein of the present invention can be made into formulations such as liquid and pulvis, be used for plant seed dressing, soak seed, water root and foliage-spray, working concentration is 10-500ug/ml, indoor pot and field test through 40 various plants, show that the mycoprotein effect is comprehensive, fungal disease, Micobial Disease, virus disease and insect pest (seeing Table 1-table 5) can not only be prevented and treated, and plant growth (seeing Table 6-table 9) can be significantly promoted; According to incompletely statistics, mycoprotein reaches 40-80% to the anti-efficient of disease and pest, and the crop yield rate reaches 4-85%.Production technique of the present invention is simple, and cost is lower, and the lasting period is long, can be widely used in efficient, the high-quality of crop, nuisanceless production and fruit fresh-keeping.
Table 1 is handled the effect that Flos Pelargonii is induced anti-grey mold fungal disease with Fungal Protein
Handle | Inoculation | Effect % |
The same day grey mold-10ug/ml grey mold-20ug/ml mould-10ug/ml mould-20ug/rml damping fluid (5mMKPO
4)
| Inoculation inoculation inoculation inoculation inoculation in the 7th day | The 13rd day 73.08 76.92 38.46 42.31 0 | The 15th day 62.86 71.43 37.14 40.00 0 | The 20th day 60.98 63.41 27.95 34.15 0 |
Annotate: the T-test shows: the level 95% and 99%, there are statistical significant difference in Fungal Protein and damping fluid between handling.
Table 2 Fungal Protein is handled the effect that tomato is induced resistance to bacterial wilt
Handle | Inoculation | Effect % |
Grey mold 20ug/ml double cross on same day pink mold 20ug/ml mould 20ug/ml damping fluid (5mMKPO
4)
| Inoculation inoculation inoculation inoculation in the 7th day | The 13rd day 76.92 61.54 38.46 0 | The 15th day 72.73 54.55 31.82 0 | The 20th day 70.97 54.84 29.03 0 |
Annotate: the T-test shows: on 95% and 99% level, have statistical significant difference between Fungal Protein and the damping fluid.
Table 3 Fungal Protein is handled the effect that Flos Pelargonii is induced anti-xanthomonas disease
Handle | Inoculation | Effect % |
The same day grey mold-10ug/ml grey mold-20ug/ml double cross pink mold-10ug/ml double cross pink mold-10ug/ml damping fluid (5mMKPO
4)
| Inoculation inoculation inoculation inoculation inoculation in the 7th day | The 13rd day 75.00 83.33 58.33 66.67 0 | The 15th day 75.00 78.57 57.14 64.29 0 | The 20th day 65.12 77.57 51.16 60.47 0 |
Annotate: the T-test shows: on 95% and 99% level, have statistical significant difference between Fungal Protein and the damping fluid.
Back 4 days evoking tobacco mosaic disease resistance effects of inoculation in the 7th day behind the table 4 spray Fungal Protein
Sample | Spraying leaf 1 TMV scab number | Spraying leaf 2 TMV scab numbers | Spraying leaf 3 TMV scab numbers | The average scab number of spraying leaf | Spraying % effect | Spraying P<0.05 | Spraying P<0.01 |
Grey mold-10ug/ml grey mold-20ug/ml double cross pink mold-10ug/ml double cross pink mold-20 ug/ml mould-10ug/ml mould-20ug/ml buffer solution (5mMKPO4)
| ?????7 ?????6 ?????9 ?????8 ?????23 ?????21 ?????64 | ????7 ????7 ????12 ????8 ????27 ????19 ????62 | ????8 ????7 ????10 ????11 ????28 ????23 ????61 | ????7.33 ????6.67 ????10.33 ????9.00 ????26.00 ????21.00 ????62.33 | 88.24 89.30 83.42 85.56 58.29 66.31 do not have | Significantly or not | Significantly or not |
Table 5 mycoprotein is to the influence of tomato virus disease and fruit disease
Handle | Virus disease morbidity number (240 strain) | Preventive effect (%) | Dead strain number | Preventive effect (%) | All kinds of fruits decayed fruit number | Preventive effect (%) |
?60ug/ml ?30ug/ml ?15ug/ml ????CK | ????0.30 ????0.67 ????2.30 ????19.30 | ??98.4
**??96.5
**??88.0
** | ????0 ????0 ????0 ????3.3 | ????100 ????100 ????100 | ??11.33 ??13.67 ??17.00 ??28.30 | ???60.1 ???51.6 ???39.9 |
Annotate: decayed fruit comprises all of scab fruits on the fruit, the disease species that causes is a kind of incessantly.
Table 6 is handled seedling altitude effect after the tomato with Fungal Protein
Handle | Efficient % | Efficient % | Efficient % |
The 1st day grey mold-10ug/ml grey mold-20ug/ml mould-10ug/ml mould-slow (5mMKPO of 20ug/ml buffering4)
| Do not have in the 14th day 9.52 11.43 6.67 6.67 | Do not have in the 18th day 10.14 12.16 6.08 7.43 | Do not have in the 21st day 10.28 13.08 6.07 7.48 |
Annotate: originally be treated to mycoprotein and soak one night of tomato seed, and in 21 °-24 ℃ growth indoor raising of seedling.The T-test shows: between Fungal Protein processing and damping fluid processing, on 95% and 99% level statistical significant difference is arranged.
Table 7 mycoprotein is to the influence of yield of hot pepper
Handle | Mean yield (jin) | Average fruit number (individual) | The average weight/really (jin) | Comparison photograph ± % (mean yield) | Comparison photograph ± % (average fruit number) |
?60ug/ml ?30ug/ml ?15ug/ml ????CK | ????49.3 ????45.3 ????42.4 ????31.1 | ????294 ????278 ????259 ????217 | ???0.072 ???0.072 ???0.072 ???0.066 | ????58.5 ????45.7 ????36.3 | ????35.5 ????28.1 ????19.4 |
The influence of table 8 pair The yield of watermelon
Handle | The real mean yield of receiving in 7/13-16 sub-district | The average melon and fruit number in 7/13-16 sub-district | Average single heavy (jin) | Comparison photograph ± % (the average actual quantities in sub-district) | Comparison photograph ± % (the average single melon in sub-district is heavy) |
??60ug/ml ??30ug/ml ??15ug/ml ?????CK | ????157.8
**????156.1
**????141.6
**??????97
| ????19.7 ????19.7 ????20 ????14 | ????8 ????7.9 ????7.4 ????4.8 | ?????66.50 ?????60.76 ?????49.02 | ?????66.67 ?????64.58 ?????54.17 |
The influence of table 9 pair tomato output
Handle | Mean yield (jin/sub-district) | Mean fruit weight (jin) | Comparison photograph ± % output | Comparison photograph ± % single fruit weight |
?60ug/ml ?30ug/ml ?15ug/ml ????CK | ????1001 ????955 ????858 ????546 | ????1.15 ????0.98 ????0.86 ????0.55 | ??????83.3 ??????74.9 ??????57.3 | ??????107.1 ??????77.4 ??????55.5 |
Table 10 mycoprotein is to the influence of young rice seedlings growth
Handle | Height of seedling (cm) (15 basins are average) | Plant dry weight (g) (6 basins are average) |
Mycoprotein 440 ug/ml CK | ????15.22
**????13.73
| ????0.32
*????0.29
|