Study On Inhibition Plant Pathogens Activity And Antifungal Protein Of Amaranthus Tricolor Water Extract

The ethyl acetate extract of Amaranthus tricolor has inhibition plant pathogens activity found in previous research.Amaranthus tricolor has the potential to be used as a source of plant source.This study further studies the inhibition plant pathogens activity of Amaranthus tricolor water extract.The aim is to isolate and purify the antifungal from Amaranthus tricolor water extract and its antifungal activity.The research will lay a foundation for cloning and separating the antifungal genes and studying the function and antifungal mechanism of Amaranthus tricolor.The contents and results of the study are as follows:1 The molecular identification of the tested Phytophthora capsici and Phytophthora melonis:collected at 20℃breeded 4d of Phytophthora capsici and Phytophthora melonis,Ezup column fungal genomic DNA extraction kit for DNA extraction,as a template,ITS universal primers ITS1:5’-TCCGTAGGTGAACCTGCGG-3’and ITS4:5’-TCCTCCGCT TATTGATATGC-3 as primers,was amplified by PCR and sequenced the amplified products.The sequence measured was matched with the NCBI logon number KM369965.1 of Phytophthora capsici and JN054403.1 Phytophthora melonis 100%.2 Extraction and activity determination of Amaranthus tricolor water extract:（1）Extraction and activity assay:weigh 50g of Amaranthus tricolor dried powder,use sterile water as the extraction solvent,and the ratio of solid to liquid 1:10,shaking at 30℃for 24h,and concentrating at 50℃to 80 mL is water extract of Amaranthus tricolor;water extract of Amaranthus tricolor is made into a drug-filled plate,and Phytophthora capsici and Phytophthora melonis are used as the test fungus.The results show that the fungistatic rate is100%and 60.08%respectively,and the fungistatic effect significantly,it showed that fungistatic substances were found in water extract of Amaranthus tricolor;（2）Extraction condition optimization:The optimum extraction conditions and orthogonal experiment L9（3⁴）determined that the optimum extraction conditions for water extract of Amaranthus tricolor were as follows:the ratio of solid to liquid was 1:10,the extraction temperature was 30℃,the oscillation time was 24h.Under this condition,the maximum extraction rate is 7.62%of water extract of Amaranthus tricolor,the inhibition rate of water extract of Amaranthus tricolor to Phytophthora capsici was 66.74%;（3）Determination of virulence:The water extract of Amaranthus tricolor was formulated into 5 different concentration gradients using a 2-fold dilution method.Phytophthora melonis and Phytophthora capsici were used as the test fungus.Metalaxyl solution for the positive control,sterile water was used as a negative control for the indoor virulence test.The EC₅₀ of water extract of Amaranthus tricolor for Phytophthora melonis and Phytophthora capsici was 110.90mg/mL and 303.39mg/mL respectively.3 Separation of water extract of Amaranthus tricolor and determination of virulence of isolates:The water extract of Amaranthus tricolor were centrifuged at 8500r/min for 30min at 4℃in a cryogenic centrifuge with a filter volume of 100 KD.Repeated 3 times and collected the filtrate is small molecular water extract of Amaranthus tricolor,and the inner liquid is water extract of Amaranthus tricolor macromolecules.The fungistatic effect of small molecular water extract of Amaranthus tricolor reached 100%.It had no fungistatic activity against Phytophthora capsici.It was not completely inactivated by small molecular water extract of Amaranthus tricolor under 100℃,and the inhibition rate against Phytophthora melonis up to 55.11%with good thermal stability;the water extract of macromolecules from Amaranthus tricolor has an fungistatic rate of 59.80%against Phytophthora capsici,at 100℃has no fungistatic activity against Phytophthora capsicii.The toxicity of Amaranthus tricolor macromolecule water extract to Phytophthora capsicii in laboratory was EC₅₀=84.52μg/mL,and the correlation coefficient r=0.9930.The leaves of pepper were inoculated in vitro.the leaves of the pepper treated with metalaxyl solution and sterile water were positive and negative.The results showed the prevention effectthat of the water extract of macromolecules from Amaranthus tricolor on Phytophthora capsici was88.8%.the therapeutic effect was 66.7%.based on macromolecules and high temperatures inactive,It was initially thought that the water extract of macromolecules of Amaranthus tricolor was a protein material（called crude protein extracted from water of Amaranthus tricolor）,The EC₅₀ value of crude protein extracted from water of Amaranthus tricolor was significantly lower than that of water extract of Amaranthus tricolor against Phytophthora capsici,and the virulence was increased.This indicated that the fungistatic substances in water extract of Amaranthus tricolor were purified and its fungistatic activity was accordingly increased.4 The basic properties and purification of crude protein from water extract of Amaranthus tricolor:（1）Determination of protein content:Bradford method was used to prepare bovine serum albumin standard curve as y=0.0095x+0.0571.It was measured with spectrophotometer at the wavelength of 595nm.The OD_595nm of crude protein from water extract of Amaranthus tricolor was 2.291,corresponding to the standard curve,crude protein from water extract of Amaranthus tricolor was 235.15μg/mL;（2）The stability of crude protein from water extract of Amaranthus tricolor was studied:Crude protein from water extract of Amaranthus tricolor was at pH=3⁹.At the time,the fungistatic activity was relatively stable,and at pH=11,its fungistatic activity was almost completely inactivated.The fungistatic activity of crude protein from water extract of Amaranthus tricolor was relatively stable below 60℃,and it was higher than 60℃.The fungistatic activity decreased with increasing temperature,and the inhibition rate was only 6.18%at 80℃.When the temperature was 100℃,crude protein from water extract of Amaranthus tricolor completely inactivated,crude protein from water extract of Amaranthus tricolor can maintain a stable fungistatic activity for 4¹6h under UV light irradiation.The fungistaticl activity didn’t change significantly with the extension of UV light irradiation time,indicating that its fungistatic activity can be short in UV light.The stability was still maintained under the irradiation of time;crude protein from water extract of Amaranthus tricolor was able to maintain stable fungistaticl activity when stored at 4℃and-20℃.（3）Morphological effects on Phytophthora capsici mycelia:Phytophthora capsici mycelia treated with crude protein from water extract of Amaranthus tricolor was observed under a microscope.The shape of the mycelium was deformed,localized swollen and expansion,and mycelia branches were increased,indicating that crude protein from water extract of Amaranthus tricolor can cause the Phytophthora capsici mycelia to deform and inhibit the normal growth of mycelia.（4）Crude protein from water extract of Amaranthus tricolor further isolation and purification:Crude protein from water extract of Amaranthus tricolor was purified by anion column chromatography using GE’s AKTA Explor chromatography system（Amersham AKTA Explore system）.The eluted protein was collected according to the value of A₂₈₀ to obtain seven elution peaks.Numbers are X1,X2,X3,X4,X5,X6,and X7.Through the indoor virulence assay,it was found that all of the 7 elution peaks had good fungistatic activity against Phytophthora capsici and lost fungistatic activity after high temperature treatment.The bands were detected by SDS-PAGE electrophoresis and mostly concentrated in the range of 25 to 116KD,which was in accordance with the molecular weight cut off of the ultrafiltration tubes.The results showed crude protein from water extract of Amaranthus tricolor was antifungal protein.5 Identification of the antifungal protein of Amaranthus tricolor:The LC-MS/MS technique was used to analyze and obtain the related information such as the charge and peak pattern of 7 eluting peaks.When the confidence conf≥95%and Unique peptides≥1,matched the elution peaks.The highest value of 3 protein sequences were AGAMOUS protein,malate dehydrogenase and amarandin-S,and the matching rates were 2.85%,64.24%,and 26.41%respectively.AGAMOUS protein has a sequence length of 246bp and a molecular weight of 28.5KD,plays an important role in plant flower organ development and is also crucial for the formation of fruits and seeds.Malate dehydrogenase protein sequence is 344bp in length.With a mass of 36.5KD,it is ubiquitous in animals,plants,bacteria and other organisms and is one of the key enzymes in the metabolism of bio-glycans.It catalyzes the reversible conversion between malic acid and oxaloacetate;the length of Amarandin-S protein sequence It is 284bp with a molecular mass of 32.0KD.Amarandin-S ribosome inactivation protein（RIP）is widely present in higher plant cells and is distributed in small numbers in fungi and bacteria.Studies have shown that RIP have a broad-spectrum antiviral activity,exhibiting a killing effect on fungi,insects,etc.,and transgenic antiviral plants have been cultivated through genetic transformation in agricultural production.