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

Abstract:【Objective】Mango (Mangifera indica L.) fruit is a popular and economically important fruitgrown 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 otherphytochemical 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 in-dustries while fungal disease and other pests can be managed at acceptable levels. It affects all aerialparts of the mango plant and can be very destructive in areas where high temperatures and rainfalls occurconcomitantly. 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 beginas 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 symptomsappear as small water-soaked spots on the lenticels. These spots later become star-shaped, erumpent andexude an infectious gum. Often, a“tear stain”infection pattern is observed on the fruit. Severe fruit infec-tions will cause premature fruit drop. Less severe infections reduce fruit quality and allow entry of otherpathogens. The mango bacterial leaf spot pathogen virulence factors include the cell wall degrading en-zymes (CWDEs) , toxins, extracellular polysaccharide and hormones, etc. The toxins are secreted by theplant pathogens outward role in plant cell walls or plant tissue. The purpose of this experiment was to ex-plore the biological activities, physical and chemical properties of the crude toxin produced by pathogensand their pathogenic effects were studied during the disease developing based on the characteristics of thepathogen with the decomposition of the host tissue. This study aims to reveal the pathogenic mechanismsof toxin in the mango bacterial leaf spot, providing the basic theory of evidence for effective disease con-trol.【Methods】‘Tainong No.1'mango leaves for the test material, the method of in vitro leaves needlescraping was used for the toxin biological activity assay. By measuring the change of mango leaves defenseenzyme, soluble protein, total sugar and chlorophyll contents and analyzing it to the mango pathogenicphysiological mechanisms. At the same time in different mediums, time, temperature and p H of the cul-ture medium conditions were optimized.【Results】The filtrate liquid which was extracted in the Watanabeculture, was not a kind of toxin of protein substance which showed the most abilities. Under the 185-800 nm wavelength ultraviolet scanning, when the toxin concentration was 35%, the absorbance at 264 nm wasthe highest, OD264 was 2.933. Using fumaric acid (FUA) and succinic acid (SUA) as the prototypes, thesamples in the 302 nm and 302 nm UV light have three yellow spots, the measured Rf value of FUA was0.361, SUA was 0.944, the samples were 0.074, 0.097 and 0.542, and the contrast (methanol) was 0. Whatwe 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 causingdamage, with the increase of crude toxin concentration, the permeability of the mango leaf after retting re-ductive monosaccharide and relative conductivity also gradually rises. The assay of biological activity ofthe liquid showed it could induce the symptoms of mango bacterial leaf spot. Studies on the physiologicaland biochemical indexes of the crude toxin, showed that mango leaves were inoculated by crude toxin. Theresults also showed that the activities of peroxidase (POD) , polyphenoloxidase (PPO) , cate-lase (CAT) andphenylalanineammonialyase (PAL) were increased in the inoculated leaves. The POD activity increasedsignificantly within 12 to 48 h after inoculation, POD reached its maximum level activity at 965.33 U·g^-1·min^-1after inoculation for 48 h, while the control, inoculation mango leaves with culture medium without patho-gen, the inoculation activity value was 1.35 times that of the control. PPO reached its maximum level activ-ity at 5 354 and 8 456 U·g-1·min-1after inoculation for 24 and 72 h, which were 1.96 and 2.20 times thatof the control. CAT and PAL reached their maximum level activity at 1 341.13 and 4.02×104U·g^-1·min^-1after 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 2.06×104U·g^-1·min^-1 after inoculation for 60 h, whichwas 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 producingtoxin were as follows: Watanabe filtrate culture, pH value 7 to 8, 24 ℃, 120 h.【Conclusion】The crude tox-in 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) .