CN104004665B - One strain can alleviate the China fir endogenetic fungus of P deficiency - Google Patents
One strain can alleviate the China fir endogenetic fungus of P deficiency Download PDFInfo
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Abstract
The present invention relates to the China fir endogenetic fungus that a strain can alleviate P deficiency.Does is described endogenetic fungus papulospora (<i>Papulospora? sp.</i>) AJ14, register preservation on May 20th, 2014 at China Committee for Culture Collection of Microorganisms's common micro-organisms center, does is deposit number CGMCC? No.9187.By applying the bacterium liquid of this bacterial strain, carrying out China fir transplanted seedling tree and dipping in root and water root for China fir forest land early stage.The bacterial strain that the present invention relates to is by the P deficiency test of plant after inoculation, show that the height of seedling leading thread of bacterial strain, biomass and chlorophyll content are all largely higher than contrast, show that it is coerced have larger function for alleviation plant phosphorus, thus confirm that obtaining this strain endogenetic fungus can promote China fir growing under P deficiency condition further.
Description
Technical field
The present invention relates to the China fir endogenetic fungus that a strain can alleviate P deficiency.
Background technology
The research of endophyte of plant started from for 19 end of the centurys, and Vogl isolates the first strain endophyte from rye grass LoliumtemulentumL. seed
[1].But the endophyte really started in large quantity research plant originates in the eighties in last century, mainly conduct a research in the vegetation of Temperate Region in China, subtropical zone and torrid areas.
Forefathers can isolate endophyte from majority of plant, therefore can infer that endophyte is ubiquitous in plant.At least belong in the gramineae farm crop of kind more than 290 at more than 80 in worldwide and found endophyte
[2].The endomycorrhiza be separated from plant at present can be divided into according to its unique function had: the plant growth-promoting bacterias such as vinelandii, solid potassium bacterium, solid phosphorus bacterium
[3-4], there is the biocontrol microorganisms of disease and insect resistance
[5-7]promote that plant is to the resistance bacterium of the repair ability of poor environment with having.
Xie Qingmei and Huang Jianhe (1985)
[8]to the morphological specificity of China fir endomycorrhiza and the qualification of mycorrhizal fungi, mainly do anatomy analysis with the symbiotic structure of Chinese fir root bark layer tissue and AM fungi and the mycelia that obtains from Rhizosphere Soil with wet screening-decantation and chlamydospore are observed.Result shows, according to chlamydosporic Morphologic Characteristics in the anatomy analysis of China fir endomycorrhiza and rhizosphere soil, determines that China fir endomycorrhiza belongs to vesicle one clump of branch type endomycorrhiza (being called for short VA mycorhiza).Further research China fir endomycorrhiza growing and the effect of resistance aspect to China fir, adequately and reasonably utilizes endomycorrhiza preparation to promote the problem of Growth of Chinese Fir, has great economic worth.Particularly in the red soil region that south China nutrient is barren, research endomycorrhiza strengthens the absorption of Phosphorus in Soil, is major issue urgently to be resolved hurrily in the China fir fast growing of current Red Soil Region of South China.In summary, the research in the endogenetic fungus of China fir only carries out identification research from root position, and Lignum seu Ramulus Cunninghamiae Lanceolatae leaf position and stem position is not all related to the research of endogenetic fungus.In addition, about the influence research of endogenetic fungus to China fir own growth also exists blank.
Summary of the invention
The object of the present invention is to provide a strain can alleviate the China fir endogenetic fungus of P deficiency.
A strain provided by the invention can alleviate the China fir endogenetic fungus of P deficiency, for papulospora (
papulosporasp.) AJ14, register preservation on May 20th, 2014 at China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNo.9187.
Separation, the purifying of this bacterium comprise:
A, material: by collecting the Root-bark of Chinese Fir of different year, stem section, leaf be divided into three parts, cleans up, be dispensed in valve bag, in 4 DEG C of Refrigerator stores with tap water;
---sterilized water embathes once---10% clorox soaks 7min---, and sterilized water embathes once for B, sterilization: 70% alcohol-pickled 30s.When the water after sample embathes for the last time is contained in the small beaker of sterilizing, at flat lining out in contrast, whether thorough with the sterilization of check sample, generate if any bacterium colony behind some skies, then show that sample surfaces sterilization is not thorough, need continue to adjust sterilization method
[9-11];
The selection of C, inoculation position: leaf tip, leaf central part and leaf base 3 process; Branch: upper, middle and lower is divided into 3 positions, wherein the 3rd position is branch stem junction; Root: be divided into the tip of a root and foundation 2 parts;
D, inoculation: cut by the explant vaccinating lancet after sterilization, be laid in media surface, cultivate and observe colony growth situation afterwards in 5-7 days in 28 DEG C of constant incubators.Rapidly, the bacterial strain that can first be produced carries out separation and purification in some bacterial strain breedings; Poor growth and the bacterial strain of comparatively small amt can extend observing time, until its growth stable after purifying.
Solid culture: use improvement Martin solid medium, formula is peptone 5.0g/L, yeast extract powder 2.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 1.0g/L, magnesium sulfate 0.5g/L, agar 14.0g/L, other is sterilized water, pH value 6.4 ± 0.2, culture temperature is 28 DEG C, training method is slat chain conveyor, and incubation time is 48h;
E, different types of endogenetic fungus access plate culture medium separation bred carry out purifying, obtain the above-mentioned China fir endogenetic fungus of single culture after 2-3 point connects purifying, switching;
Its asexual spore lacks, and mycelia light color, produces little cell tight knot and become the spherical of class sclerotium structure, black; With reference to Fungal identification handbook be accredited as Papulospora (
papulosporasp.).
Above-mentioned a kind of can alleviate P deficiency the preparation method of China fir endogenetic fungus application bacterium liquid, by above-mentioned bacterial strain access liquid nutrient medium, shaking table shaking culture, culture temperature is 28 DEG C, incubation time 48 ~ 72h, utilize blood counting chamber to calculate bacterial concentration, bacterium liquid ultrapure water is diluted to 1.0-9.0 × 10
6it is for subsequent use that cfu/ml is finished product; Liquid nutrient medium: be improvement Martin substratum, wherein peptone 5.0g/L, yeast extract powder 2.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 1.0g/L, magnesium sulfate 0.5g/L, other is sterilized water, pH value 6.4 ± 0.2.
Above-mentioned a kind of can alleviate P deficiency the application method of China fir endogenetic fungus, be the bacterium liquid of this bacterial strain of application, China fir transplanted seedling tree dips in root and waters root for China fir forest land early stage.
Above-mentioned a kind of can alleviate P deficiency the application method of China fir endogenetic fungus, be with 9.0 × 10
5cfu/ml bacterium liquid, to water the rhizosphere in every strain China fir by every strain 100ml in forest land.
Above-mentioned a kind of can alleviate P deficiency the application method of China fir endogenetic fungus, be the bacterium liquid 5.0 × 10 of this bacterial strain of application
5seedling is dipped in root 10min by cfu/ml before China fir Rooted Cuttings is planted.
The bacterial strain that the present invention relates to separation and purification from China fir plant obtains, through being inoculated in China fir tissue cultured seedling, according to photosynthetic indices Comprehensive Evaluation, further confirmation its to plant strain growth have alleviate P deficiency effect, search out promoting that the useful function endogenetic fungus of plant strain growth can be applicable to produce.By inoculating the P deficiency test of rear plant, show that the height of seedling leading thread of bacterial strain, biomass and chlorophyll content are all largely higher than contrast, show that it is coerced have larger function for alleviation plant phosphorus, thus confirm that obtaining this strain endogenetic fungus can promote China fir growing under P deficiency condition further.
Accompanying drawing explanation
Fig. 1 is that different phosphate coerces lower AJ14 bacterial strain to the impact of China Fir Seedling height of seedling.
Fig. 2 is that different phosphate coerces lower AJ14 bacterial strain to the impact of China Fir Seedling leading thread.
Fig. 3 is that different phosphate coerces the impact of lower AJ14 on China Fir Seedling biomass.
Fig. 4 is that different phosphate coerces the impact of lower AJ14 on China Fir Seedling chlorophyll content.
Embodiment
One strain can alleviate the China fir endogenetic fungus of P deficiency, bacterial classification called after AJ14, Classification And Nomenclature: papulospora (
papulosporasp.), its deposit number: CGMCCNo.9187, preservation date: on May 20th, 2014, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Application in 1 water planting breeding
Get the application bacterium liquid of the above-mentioned endogenetic fungus prepared, make 5.0 × 10
5cfu/ml bacterium liquid, dips in root 10min before China fir Rooted Cuttings is planted.Surviving rate more than 10% of planting seedlings can be improved.
2 rhizosphere soils water executes application
This test adopts earth culture pot experiment, tests the China fir tissue cultured seedling that China Fir Seedling used provides for forest-science academy of Fujian Province.The consistent nursery stock of growing way is selected to be colonizated in diameter 15cm, in the plastic tub of high 10cm in May, 2013.Soil used is strictly through the yellow soil of fumigation.Through weighing, the 3kg soil of equivalent put into by every basin.Through bimestrial restorative growth, continuous three days (July 5, July 6 and July 7) applies the 100mL bacterium liquid of same concentrations respectively at China fir rhizosphere (R) and tree crown (G).Totally 5 dominant bacterias utilizing above-mentioned test to obtain, 4 repetitions of often kind of bacterium liquid, with distilled water as blank.Inoculate the mensuration of carrying out indices after 60 days.
Bacterium solution preparation: liquid nutrient medium strains tested being accessed 50mL, through the cultivation of 72h in constant-temperature shaking incubator.Press decimal dilution method by stroke-physiological saline solution bacterium liquid is diluted, utilize blood counting chamber to calculate bacterial concentration and be mixed with 5.5 × 10
6cfu/mL.
2.1 height of seedling leading threads measure
Steel ruler measures height of seedling, and electronic digital indicator measures leading thread.
2.4 biomass estimation
By the different sites of plant respectively label put into the aluminium box of known weight (W1), put into 105 DEG C, baking oven and dry half an hour (deactivating enzyme), dry at 70 DEG C, after 10h, taking-up aluminium box is put into loft drier and is cooled to room temperature, put into baking oven again after weighing, replication is till constant weight.Claim to obtain the gross weight (W2) of aluminium box and sample, then sample dry weight is W2-W1.
In this test, root/shoot ratio index adopts the mass ratio of plant dry-matter.Below ground, mainly root system are underground part; More than ground, mainly stem and leaf is over-ground part.
2.2 measuring chlorophyll content
Measuring chlorophyll content is with reference to Zhang Xianzheng (1986)
[12]method, adopt acetone ethanol extraction method.Get close positions blade, clean and after suck dry moisture, accurately take the fresh leaf of 0.1g and put into small test tube, with mixed solution (acetone: ethanol=1:1) 10mL capping plug.At room temperature dark place goes clear liquid under 663nm and 645nm, to measure absorbance value respectively after extracting 12h.
Result calculates:
2.3 data processing
Data preparation and preliminary treatment adopt Excel2003, and mapping adopts Origin8.0, and the statistical analysis of data adopts SPSS18.0.
3 results and analysis
3.1 different phosphate coerce the impact of lower bacterial strain on China Fir Seedling height of seedling leading thread
Height of seedling is the index showing plant-growth the most intuitively, and seedling generally adopts leading thread to replace the diameter of a cross-section of a tree trunk 1.3 meters above the ground.
As shown in Figure 1, different phosphate coerces the impact of lower bacterial strain on China Fir Seedling height of seedling: under four different phosphate coerce gradient, under being respectively normal condition, mild stress conditions, moderate stress conditions, severe water stress condition, seedling height of seedling overall growth rate during coercing of inoculation AJ14 bacterial strain is respectively 22.69%, 20.17%, 17.49%, 17.79%.Corresponding, control group height of seedling is coerced period and is respectively 8.97%, 4.10%, 3.80%, 2.79% whole respectively coercing the overall growth rate under gradient.
As shown in Figure 2, different phosphate coerces the impact of lower bacterial strain on China Fir Seedling leading thread: under four different phosphate coerce gradient, under being respectively normal condition, mild stress conditions, moderate stress conditions, severe water stress condition, seedling leading thread overall growth rate during coercing of inoculation AJ14 bacterial strain is respectively 16.91%, 13.60%, 11.94%, 16.14%.Corresponding, control group leading thread is coerced period and is respectively 8.96%, 8.90%, 6.33%, 3.37% whole respectively coercing the overall growth rate under gradient.
3.2 different phosphate coerce the impact of lower different strain on China Fir Seedling biomass
As shown in Figure 3, coerce gradient four different phosphate, namely under normal condition, mild stress conditions, moderate stress conditions, severe water stress condition, the Seedling Biomass of inoculation AJ14 bacterial strain than contrast respectively high by 21.68%, 10.27%, 32.04%, 11.24%.
3.3 different phosphate coerce the impact of lower different strain on China Fir Seedling chlorophyll content
Under normal condition, the chlorophyll content of inoculating strain AJ14 seedling coerce to 15d, 30d, 45d and 60d respectively higher by 165.24% than contrast content, 32.45%, 22.79%, 27.01%.Under mild stress conditions, the chlorophyll content of inoculating strain AJ14 seedling coerce to 15d, 30d, 45d and 60d respectively higher by 109.52% than contrast content, 56.68%, 21.21%, 27.50%.Under moderate stress conditions, the chlorophyll content of inoculating strain AJ14 seedling coerce to 15d, 30d, 45d and 60d respectively higher by 106.47% than contrast content, 31.49%, 5.14%, 28.59%.Under severe water stress condition, the chlorophyll content of inoculating strain AJ14 seedling coerce to 15d, 30d, 45d and 60d respectively higher by 69.21% than contrast content, 24.58%, 6.76%, 22.31%.
The present invention finds the endogenetic fungus that a strain can be alleviated Phosphorus In Chinese Fir and coerces, and adopts the method for watering root to be inoculated in China Fir Seedling this strain China fir endogenetic fungal bacterial strain.By inoculating the P deficiency test of rear plant, show that the height of seedling leading thread of bacterial strain, biomass and chlorophyll content are all largely higher than contrast, show that it is coerced have very large function for alleviation plant phosphorus, thus confirm that obtaining this strain endogenetic fungus can promote China fir growing under P deficiency condition further.
reference
[1] Guo Liangdong. Recent Research Advance in Endophytic Fungi [J]. fungus system, 2001,20 (1): 148 ~ 152.
[2] in Hanshou, Ji Yanling, Weng Zhonghe etc. the biological characteristics in seedling stage [J] of grass endogenetic fungus research 10--endogenetic fungus symbiote. Practaculture Science, 2009,26 (10): 150-154.
[3]LuH,ZouWX,MengJC,HuJ,TanRX.NewbioactivemetabolitesproducedbyColletotrichumsp.,anendophyticfungusinArtemisiaannua[J].PlantSci,2000,151:67-73.
[4]ReisVM,BaldaniVLD,DobereinerJ.BiologicaldinitrogenfixationinCramineaeandpalmtrees[J].CriticalRevPlantSci,2000,19:227-247.
[5] StoneJK, BaconCE, WhiteJFJr.Anoverviewofendophyticmicrobes:endophytismdefi nedMicrobialEndophytes [J] .NewYork:MarcelDekker, 2000; 3-29. monarch Feng Yong, Song Wei. endophytic bacterium [J]. Nature Journal, 2001,23 (5): 249 ~ 252.
[6] Feng Yongjun, Song Wei. endophytic bacterium [J]. Nature Journal, 2001,23 (5): 249 ~ 252.
[7] Yang Hailian, Sun Xiaolu, Song Wei. the research [J] of endophytic bacterium. microbiology is circulated a notice of, and 1998,25 (4): 224 ~ 227.
[8] Xie Qingmei, Huang Jianhe. the morphological specificity of the research I. endomycorrhiza of China fir endomycorrhiza and the qualification [J] of mycorrhizal fungi. Fujian rice wine, 1985,5 (2): 82-84
[9] separation of Cui Lin, Sun Zhen, Sun Fu, etal. potato endogenetic bacteria and the Screening and Identification [J] of ring rot Antagonistic Fungi. Plant Pathology, 2003,33 (004): 353-358.
[10] separation of He Hong, Cai Xueqing, Hong Yongcong, etal. capsicum endogenetic bacteria and the screening [J] of Antagonistic Fungi. Chinese biological is prevented and treated, and 2002,18 (4): 171-175.
[11] Yuan Jun, Sun Fu, Tian Hongxian, etal. prevent and treat the Isolation and screening [J] of the useful endogenetic bacteria of bacterial ring rot o potato. microorganism journal, 2002,42 (3): 270-274.
[12] Zhang Xianzheng. chlorophyll content of plant measures---acetone ethanol hybrid system [J]. Liaoning agricultural sciences, 1986 (3): 26-28.
Claims (3)
1. a strain can alleviate the China fir endogenetic fungus of P deficiency, it is characterized in that: described endogenetic fungus be papulospora (
papulosporasp.) AJ14, register preservation on May 20th, 2014 at China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNo.9187.
2. an application bacterium liquid for China fir endogenetic fungus as claimed in claim 1, is characterized in that: the preparation method of described application bacterium liquid, be by described papulospora (
papulosporasp.) AJ14 accesses liquid nutrient medium, shaking table shaking culture, culture temperature is 28 DEG C, incubation time 48 ~ 72h, utilizes blood counting chamber to calculate bacterial concentration, bacterium liquid ultrapure water is diluted to 1.0-9.0 × 10
6cfu/ml, for subsequent use; Liquid nutrient medium: be improvement Martin substratum, wherein peptone 5.0g/L, yeast extract powder 2.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 1.0g/L, magnesium sulfate 0.5g/L, other is sterilized water, pH value 6.4 ± 0.2.
3. a purposes for China fir endogenetic fungus as claimed in claim 1, is characterized in that: the bacterium liquid applying this bacterial strain, and China fir transplanted seedling tree dips in root and waters root for China fir forest land early stage.
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