| Okra(Abelmoschus esculentus L.Moench)is a vegetable with high nutritional and medicinal value.However,after harvesting,okra is susceptible to infection by pathogenic microorganisms,leading to rot and deterioration.Additionally,the tender fruit pods gradually undergo lignification during storage,causing them to lose their edible value.In order to improve the post-harvest quality of okra,measures must be found to counteract the microbial infestation of okra and to regulate the lignin synthesis process in okra.In this study,Colletotrichum fioriniae and Mucor circinelloides,which were isolated from rotting okra,were used as test materials to investigate their biological characteristics.The inhibitory effects of thymol polysaccharide composite coating(TKL coating)on the two pathogenic bacteria were analyzed,and the antimicrobial mechanism was explored.Finally,the physiological effects of the coating on fresh okra pods were analyzed,including surface disease status and the impact on lignin synthesis.The main research results are as follows:1.Isolation,purification and identification of potentially pathogenic bacteriaEight fungi that may cause okra diseases were isolated and purified from naturally decaying okra and identified by 5.8s r RNA,and the eight fungi were obtained as follows:Pseudogymnoascus pannorum,Cladosporium sp,Arthrinium sp,Colletotrichum fioriniae,Hypoxylon aveirens,Fusarium fujikuroi,Aspergillus sydowii and Mucor circinelloides.The eight pathogenic fungi were inoculated onto the surface of healthy okra using the smear method.Colletotrichum fioriniae caused significant anthracnose symptoms on okra and Mucor circinelloides caused severe soft rot symptoms on severe okra;both were used as indicator fungi for post-harvest disease control of okra.Further observations were verified using the puncture inoculation method,and consistent results were obtained.2.Biological properties of two indicator fungiWhen studying the biological characteristics of the two indicator fungi,mannitol,sucrose and starch were the best carbon sources for Colletotrichum fioriniae;potassium nitrate was the best nitrogen source.p H=7 was the fastest growth of Colletotrichum fioriniae;the largest colony diameter and the best growth were achieved at a Na Cl concentration of 0.5%.The best growth temperature was at 28 ℃;lighting was favorable for its growth.Maltose was the best carbon source for Mucor circinelloides,and beef paste and potassium nitrate were the best nitrogen sources.The optimum growth p H was 6,and Mucor circinelloidesweres were suitable for growth under weakly acidic conditions.The optimum growth temperature was 28 ℃;in addition,lighting and low concentration of Na Cl were beneficial to the growth and reproduction of Mucor circinelloides.3.In vitro inhibitory effect and mechanism of TKL coating solution on two pathogenic fungi of okraIn vitro inhibition experiments revealed that;the half-effective and minimum inhibitory concentrations of TKL were 98.70 mg/L and 1000 mg/L for Colletotrichum fioriniae,113.55 mg/L and 960 mg/L for Mucor circinelloides.Concerning the two fungi’s half-effective inhibitory concentration,the thymol concentration in the subsequent TKL coating was set to 100 mg/L for both(TKL100).It was observed under scanning electron microscope that after TKL100 treatment;the mycelium of both Colletotrichum fioriniae and Mucor circinelloides would shrink significantly;the surface was rough and curled,and even obvious fracture appeared;the spores of Mucor circinelloides would also become significantly smaller and appear rough and wrinkled.After treatment with TKL100,the malondialdehyde content related to cell membrane damage caused by Colletotrichum fioriniae and Mucor circinelloides was significantly reduced,indicating that TKL100 caused damage to the cell membranes.TKL100 may have accelerated the metabolic process of Colletotrichum fioriniae,showing 1.43 times higher polygalacturonic acid enzyme activity at 48 hours,1.22 times higher cellulase activity at96 hours,and 1.33 times higher polymethylgalacturonic acid enzyme activity at 120 hours compared to the control group,achieving a antifungal effect.However,after treatment with TKL100,the polygalacturonic acid and cellulase activity of Mucor circinelloides was reduced,and the polymethylgalacturonic acid enzyme activity was first decreased and then increased.Therefore,TKL100 may inhibit the metabolism of Mucor circinelloides and the production of its metabolites,thereby achieving a antifungal effect.After induction with TKL100,chitinase and β-1,3-glucanase related to cell wall degradation of Colletotrichum fioriniae showed an increasing trend,while the chitinase activity of Mucor circinelloides continued to increase,and the β-1,3-glucanase activity showed irregular changes.Therefore,TKL has a good antifungal effect on both Colletotrichum fioriniae and Mucor circinelloides,and its main antifungal mechanisms are related to damaging cell membranes,degrading cell walls,inhibiting metabolic activity,and reducing metabolites.4.Effects of TKL coating on post-harvest physiological metabolism of okraFrom observations of surface diseases and hardness tests,it can be seen that TKL100 can inhibit to a certain extent the infection of okra by Colletotrichum fioriniae and Mucor circinelloides,slow down the appearance of corresponding disease symptoms on the surface of okra,reduce the incidence of brown spots,wrinkles,and white mycelium formation,and inhibit the softening of okra tissue.The mechanism of TKL100 in preventing anthracnose caused by Colletotrichum fioriniae is related to the regulation of okra lignin synthesis pathway to some extent.It promotes the synthesis of lignin precursors by stimulating the activity of key enzymes in the lignin synthesis pathway,and the total phenols and total flavonoids content of the coating group at 10 days was the highest,which were 10.41 ± 0.17 mg GAE/g DW and 3.84 ± 0.56 mg RE/g DW,respectively.The PAL,C4 H,and 4CL activities of the coating group were highest at 10 days,which were 624.77 ±8.24 U(h.g),925.69 ± 32.10 U/mg FW,and 2177.43 ± 1.95 U/L,respectively,achieving good anti-stress and disease prevention effects.However,the mechanism of TKL100 in preventing soft rot caused by Mucor circinelloides is not yet determined to be related to okra lignin synthesis pathway.It cannot be ruled out that after TKL treatment,PAL,which is a key precursor enzyme in okra lignin synthesis,also showed a significant increase,but further experiments are needed to verify this in the later stage. |
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