Biological activity, physical and chemical properties and pathogenic effect of crude toxic produced by pathogen causing mango bacterial leaf spot

ZHANG Dazhi; ZHAN Rulin; LIU Feng; ZHAO Yanlong; HE Yanbiao; CHANG Jinmei

doi:10.13925/j.cnki.gsxb.20160452

Abstract：[Objective] Mango (Mangifera indica L.) fruit is a popular and economically important fruit grown worldwide throughout the tropics and subtropics,due to its excellent qualities such as bright color,sweet taste and luscious flavor,and nutritional composition such as vitamins,minerals,fiber,and other phytochemical compounds.Mango bacterial leaf spot,a bacterial disease caused by Xanthomonas campestris pv.Mangiferaeindicae,is very difficult to control,and it usually becomes a limiting factor for mango industries while fungal disease and other pests can be managed at acceptable levels.It affects all aerial parts of the mango plant and can be very destructive in areas where high temperatures and rainfalls occur concomitantly.The bacterial pathogen can infect wounds and natural openings.Leaf and fruit symptomsare most common,but twig and branch cankers may occur when infection is severe.Leaf symptoms begin as small water-soaked spots delineated by veins,becoming raised,black,sometimes with a chlorotic halo.Several months after infection,leaf lesions dry and turn light brown ash-gray.In addition,fruit symptoms appear as small water-soaked spots on the lenticels.These spots later become star-shaped,erumpent and exude an infectious gum.Often,a "tear stain" infection pattern is observed on the fruit.Severe fruit infections will cause premature fruit drop.Less severe infections reduce fruit quality and allow entry of other pathogens.The mango bacterial leaf spot pathogen virulence factors include the cell wall degrading enzymes (CWDEs),toxins,extracellular polysaccharide and hormones,etc.The toxins are secreted by the plant pathogens outward role in plant cell walls or plant tissue.The purpose of this experiment was to explore the biological activities,physical and chemical properties of the crude toxin produced by pathogens and their pathogenic effects were studied during the disease developing based on the characteristics of the pathogen with the decomposition of the host tissue.This study aims to reveal the pathogenic mechanisms of toxin in the mango bacterial leaf spot,providing the basic theory of evidence for effective disease control.[Methods] ‘Tainong No.1’ mango leaves for the test material,the method of in vitro leaves needle scraping was used for the toxin biological activity assay.By measuring the change of mango leaves defense enzyme,soluble protein,total sugar and chlorophyll contents and analyzing it to the mango pathogenic physiological mechanisms.At the same time in different mediums,time,temperature and pH of the culture medium conditions were optimized.[Results] The filtrate liquid which was extracted in the Watanabe culture,was not a kind of toxin of protein substance which showed the most abilities.Under the 185-800nm wavelength ultraviolet scanning,when the toxin concentration was 35％,the absorbance at 264 nm was the highest,OD264 was 2.933.Using fumaric acid (FUA) and succinic acid (SUA) as the prototypes,the samples in the 302 nm and 302 nm UV light have three yellow spots,the measured Rf value of FUA was 0.361,SUA was 0.944,the samples were 0.074,0.097 and 0.542,and the contrast (methanol) was 0.What we learn from this is that the toxins have more than three types of organic acids,but not FUA and SUA.The mango bacterial leaf spot pathogen secretes crude toxin on the mango leaf cell membrane causing damage,with the increase of crude toxin concentration,the permeability of the mango leaf after retting reductive monosaccharide and relative conductivity also gradually rises.The assay of biological activity of the liquid showed it could induce the symptoms of mango bacterial leaf spot.Studies on the physiological and biochemical indexes of the crude toxin,showed that mango leaves were inoculated by crude toxin.The results also showed that the activities of peroxidase (POD),polyphenoloxidase (PPO),cate-lase (CAT) and phenylalanineammonialyase (PAL) were increased in the inoculated leaves.The POD activity increased significantly within 12 to 48 h after inoculation,POD reached its maximum level activity at 965.33 U·g-1 ·min-1 after inoculation for 48 h,while the control,inoculation mango leaves with culture medium without pathogen,the inoculation activity value was 1.35 times that of the control.PPO reached its maximum level activity at 5 354 and 8 456 U·g-1 ·min-1 after inoculation for 24 and 72 h,which were 1.96 and 2.20 times that of the control.CAT and PAL reached their maximum level activity at 1 341.13 and 4.02×104 U· g-1· min-1 after inoculation for 36 h,which were 1.08 and 4.19 times that of the control.After being sharply lower,PAL again reached its maximum level activity at 2.06× 104 U·g 1· min-1 after inoculation for 60 h,which was 2.84 times that of the control.And the soluble protein and sugar content were significantly increased,while the content of chlorophyll decreased.After shaking the culture,the optimum conditions of producing toxin were as follows:Watanabe filtrate culture,pH value 7 to 8,24 ℃,120 h.[Conclusion]The crude toxin obviously produced retting on the mango leaves,which shows that it can change cell membrane permeability,lead to the organization of mango leaf electrolyte leakage,and cause the damage effects of blade cell membrane,eventually giving rise to the occurrence of diseases.The results indicate that the crude toxin is maybe one of the pathogenicity factors of mango bacterial leaf spot pathogen,which may belong to non-host specific toxins (NHST).