杧果细菌性角斑病菌细胞壁降解酶的致病作用

Pathogenic effect of cell wall degrading enzymes produced by pathogen causing mango bacterial leaf spot

【目的】从细胞壁降解酶的角度开展其致病机制的研究,探讨杧果细菌性角斑病在发病过程中产生细胞壁降解酶的种类及其在致病过程中的作用,以期为对该病害的有效监测和研发防控新技术提供理论依据。【方法】采用活体外诱导培养和病原菌接种处理,利用3,5-二硝基水杨酸法（DNS法）和紫外分光光度计法检测病原菌侵染过程中细胞壁降解酶活性变化,DDS-ⅡA型电导率仪测定叶片组织浸出液的电导率,并观测细胞壁降解酶对杧果叶片的致病作用。【结果】病原菌在改良的Marcus培养液和罹病组织中均能产生6种主要细胞壁降解酶,其中羧甲基纤维素酶（Cx）活性较高,多聚半乳糖醛酸酶（PG）、果胶甲基半乳糖醛酸酶（PMG）和β-葡萄糖苷酶次之,果胶甲基反式消除酶（PMTE）和多聚半乳糖醛酸反式消除酶（PGTE）较低。病原菌接种杧果叶片后,6种酶活性显著升高,在病菌侵染前期（0-4 d）,PMG接种后2 d达到最大活性高峰,酶活值为8.470 U·mg^-1,以未含致病菌株的培养基接种杧果叶片作为对照,此时接种处理酶活值是对照的3.805倍;β-葡萄糖苷酶接种后4 d酶活性值最大,为15.190 U·mg^-1,是对照的4.388倍。在病菌侵染后期（4-14 d）,Cx、PG、PMTE和PGTE均在接种后的10 d达到最高峰,酶活性值分别为25.941、14.605、0.009、0.014 U·mg^-1,是对照的2.672、14.634、12.571和4.121倍,在整个测定过程中,对照始终处于较低水平。此外,细胞壁降解酶对杧果叶片具有浸解作用,且浸解损伤程度与酶浓度成正比。【结论】细胞壁降解酶在病原菌的入侵和致病过程中起重要作用。果胶酶在病原菌侵染初期最早分泌并起作用,纤维素酶主要降解次生壁,致病作用发生在后期,并且果胶酶比纤维素酶对杧果叶片的致病作用明显。

【Objective】Mango（Mangifera indaca L.） fruit is cultivated throughout the tropics and also in subtropical areas such as Israel, North Africa, Spain, Florida and Japan, and ranks the fifth in production worldwide. Major producers are India, China, Mexico, Thailand, Pakistan, and the Philippines. Mango bacterial leaf spot, a bacterial disease caused by Xanthomonas campestris pv. mangiferaeindicae, can be very destructive in areas where high temperatures and rainfalls occur concomitantly, and seriously affects mango yield and fruit commodity value. It is one of the main diseases in mango under quarantine check. The disease results in abnormal decomposition in infected leaves and fruit. It also causes black cross-crack spots on the surface of the infected tissue. The infected parts in the leaves initially develop water-soaked brown yellow halo spots which gradually develop into large black spots. In the infected branches, the adhesive material from crap gap faded out, and green evolved into black gradually. Dark green star spots emerge early and turn black later with a large number of bacteria that spilled out of the infected site of fruit from the middle crack, which formed slightly viscous strip blotch on its surface, and the fruit with serious disease proned to drop. The disease fruit was got, that aggravated the spread of the epidemic in transit easily. Pathological mechanisms of the mango bacterial leaf spot include cell wall degrading enzymes（CWDEs）, toxic extracellular polysaccharides and hormones, etc. The CWDEs are the enzymes secreted by the plant pathogens into plant cell walls. The objective of the study was to explore the cell wall degradation enzymes produced by pathogens, and their pathogenic effect were studied during the development of the disease based on the characteristics of the decomposition of the host tissue by the pathogen. The study aims to clarify the epidemic characters of the disease and to provide the theoretical basis for effective disease control.【Methods】This research was conducted using in vitro culture and pathogen inoculation. The CWDEs were detected by using 3,5- dinitrosalicylic acid method（DNS method） and UV spectrophotometer, and the DDS-ⅡA type conductivity meter was used to measure the membrane leaching in the leaves.The pathogenic effect of CWDEs on mango leaves was also observed.【Results】The CWDEs were produced in an improved Marcus′s medium and in the diseased leaves infected by Xanthomonas campestris pv. mangiferaeindicae. Among the six CWDEs, carboxymethyl cellulase（Cx） was the highest, followed in an descending order by polygalacturonase（PG）, pectin methylgalactuionase（PMG）, β-glucosidase, polygalacturonic acid transeliminase（PGTE） and pectin methyltrans- eliminase（PMTE）. In the diseased leaves, high enzyme activities were detected at the interface between the healthy and diseased parts, and activities of Cx, β-glucosidase, PG and PMG were significantly higher than those of PMTE and PGTE.However, all these six enzymes increased significantly after the invasion of the pathogen. In the early stage of invasion（0 to 4thdays）, PMG reached its maximum activity of 8.470 U·mg^-1 2 days after inoculation. Pathogen inoculation caused 3.805 times higher in PMG activity than the control, where leaves were cultured in a medium without pathogen. β-glucosidase reached its maximum level activity of 15.190 U·mg^-1 4 days after inoculation, which was 4.388 times of the control. In the later stage of invasion（4 to 14 th days）, Cx, PG, PMTE and PGTE reached their maximum activities of 25.941, 14.605, 0.009 and 0.014 U·mg^-1 10 days after inoculation, respectively, which was 2.672, 14.634,12.571 and 4.121 times of the control, respectively. Throughout the experiment, enzyme activities in the control always maintained low. After culturing for 6 days, the leaves treated with extracted and purified pectinase produced significantly larger disease spots than those treated with cellulase, and the control produced no obvious disease spots.The diseased spots on the leaves secreted CWDEs into leaf cells with damaged membrane, and the reducing sugar content and relative conductivity of leachate from mango leaf tissues increased gradually with the increasing concentrations of CWDEs.【Conclusion】Cellulase and pectinase were produced by the pathogen, which played an important role in lesion formation and expansion. PMG and β- glucosidase reached their maximum activities in the early stage of pathogen attack, but PMG appeared earlier. Cx and PG reached their maximum levels later, 10 days after inoculation, but the activity of Cx was higher. Plant cell walls are made up of the middle layer, the primary wall and secondary wall. The middle layer is mainly composed of pectic substances. It could clearly be seen pectinase from the pathogen was the earliest in secretion and action, and cellulase mainly acted upon the secondary wall, while pathogenic effect occurred later. Therefore the pathogenic effect of pectinase was more significant than cellulase on mango leaves.