Lili ZHANG, Youhong CHANG, Zhiyi CHEN

1. Agro-technical Station of Xiangcheng Agricultural Bureau of Suzhou City, Suzhou 215000, China;

2. Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;

3. Institute of Horticulture, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China

Physalospora piricola Nose is caused by Botryosphaeria berengeriana f. sp. piricola,this pathogenic bacteria mainly infects branches and fruits of pears, and also infects leaves. Botryosphaeria berengeriana f. sp. piricola has wide host range, except to pears, it mainly hazards to apples, peaches, apricots,Malus asiatica, hawthorn, jujube and other fruit trees. When the pathogenic bacteria harmed to fruits,the fruits became rotten and with the form of Physalospora, and the output yearly decreased about 25%, meanwhile the rotten fruit rate reached 80% above in serious case. Bacillus subtilis is a kind of Bacillus. By means of successfully colonizing to the rhizosphere, the surface or in vivo of plants, it competedwith pathogenic bacteria for the surrounding nutrition, secreted antimicrobial substances to inhibit the growth of pathogenic bacteria, and induced defense mechanism of pears to against pathogenic bacteria invading so as to achieve the purpose of biological control[1]. The reports about the biological control of Bacillus subtilis for Botryosphaeria berengeriana f. sp.piricola were more. Zhao Baige et al.[2]indicated that Bacillus subtilis B-903 had better inhibiting effect on Botryosphaeria berengeriana f. sp.piricola in vitro or in fruits. Xie Dong et al.[3]indicated that the isolated Bacillus subtilis BS-98 could inhibit Physalospora piricola and other pathogenic fungi of plants. In addition, the applications of Bacillus subtilis in biocontrol of plant diseases had been reported[4].This study do the research of the change rule of antioxidant enzymes system of the fruits by inoculating Botryosphaeria berengeriana f. sp.piricola and spraying Biocontrol bacteria to pears,in order to provides a theoretical basis for further understand defense mechanism of pear after inoculated Botryosphaeria berengeriana f. sp. piricola and the enzyme activity mechanism of Biocontrol bacteria.

Materials and Methods

Materials

Botryosphaeria berengeriana f.sp. piricola was provided by Jiangsu Academy of Agricultural Science biocontrol group, used after activation cultured on PDA culture medium.Biocontrol bacteria sf-19(Bacillus subtilis)was provided by Jiangsu Academy of Agricultural Science institute of Plant Protection biocontrol group.The variety of test pear was "Golden Twentieth Century" collected from Jiangsu Academy of Agricultural Sciences Institute of Horticulture, which was ripe, uniform size, healthy and fresh.

Experimental design

The middle and upper part of each pear evenly drilled two holes,then treated as follows: ①CK group treated by sterile; ②S group treated by Biocontrol bacteria; ③ L group treated by Botryosphaeria berengeriana f.sp.piricola;④L+S group treated by Botryosphaeria berengeriana f.sp. piricola and then sprayed Biocontrol bacteria.

Enzyme extraction and enzymatic activity determination

2 g of pear pulp around the inoculation position was collected at 2, 6,12, 24, 48 h after inoculated respectively. Then put into a mortar , added 10 ml of 0.05 m/L phosphate buffer(pH7.8)and a little silica sand,grind up to homogenate in ice bath,centrifuged at 10 000 r/min for 20 min at 4 ℃.The supernatant was used to detemine the indicators.Enzyme activity determined by 9100 UV spectrophotometer. The determination methods of the content of MDA and the enzyme activity of POD and SOD referred to Li heshen[5].The determination methods of the enzyme activity of CAT referred to Zhu meiyun[6], which one enzyme activity unit (U)referred to △OD240decreased 0.01 units within 1min. The determination methods of the enzyme activity of PPO referred to Tan xingjie[7],which one enzyme activity unit(U)referred to△OD420decreased 0.01 units within 1 min. Different times after inoculation was the abscissa, and the determined enzyme activity was the ordinate.

Results and Analysis

Dynamic changes of MDA content

When the plant organs during aging or under stress conditions, the cell membranes suffered different degrees of damage, so the membrane permeability increased and led to intracellular electrolyte leakage. Meanwhile, reactive oxygen species in the cells largely accumulated, which always occurred membrane lipid peroxidation. MDA is one of its products, which was often use as the index of membrane lipid peroxidation and reflected the degree of membrane lipid peroxidation and the strength of the stress response of plants. The results indicated that four treatment groups had a certain influence on MDA content in pear fruits(Fig.1). The content of MDA of CK treatment was lower and the variation trend was moderate. The content of MDA treated by Biocontrol bacteria(S)had little difference with CK, which indicated that the biocontrol bacteria had little effect on MDA content. The content of MDA treated by Botryosphaeria berengeriana f.sp.piricola(L)gradually increased from 2h to 48h, and reached high peak in 48 h,was 10. 22 nmol/g which was 1.86 times of CK. It indicated that the Botryosphaeria berengeriana f. sp. piricola had great effect on MDA content. The content of MDA treated by Botryosphaeria berengeriana f. sp. piricola and biocontrol bacteria increased rapidly from 6 h to 24 h,reached high peak in 24 h, was 8.92 nmol/g which was 1.62 times of CK. It indicated that the Botryosphaeria berengeriana f. sp.piricola and biocontro bacteria (L+S)had greater effect on MDA content,but decreased from 24 h to 48 h, which means that biocontrol bacteria might play a inhibiting effect.

Dynamic changes of SOD, CAT,POD activity

SOD commonly exists in plants and animals, which is a enzyme that can eliminate superoxide anion free radical O2-.CAT commonly exists in all tissues of plants, and its activity has a certain relationship with the strength of metabolism, cold resistance and disease resistance of plants. POD also commonly exists in plants,it has higher activity, and its activity change can reflect the change of metabolism of plants in certain period,which is one of the key enzymes of reactive oxygen defense system. CAT, POD and SOD form an effective active oxygen scavenging system, which cooperate harmoniously with each other and effectively eliminate free radicals and peroxides. Within a certain range, SOD and CAT transformed O2-and H2O2into H2O and O2, and decreased the formation of -OH which was toxic and highly active. POD and CAT can catalyze H2O2to form H2O,so as to effectively prevent the accumulation of H2O2and O2-,and restricted these free radicals to activated membrane lipid peroxidation[8]. In addition, POD can catalyze lignin monomers polymerize to synthesize lignin under the participation of H2O2, which improved the degree of lignification of tissues, thereby prevented the invasion of pathogenic bacteria.

The results showed that the enzyme activity of SOD of CK in 48 h had little different with 2 h. The enzyme activity of SOD treated by Biocontrol bacteria (S) and Botryosphaeria bere-ngeriana f. sp. piricola (L) in 2 h had little different with 12h, and Botryosphaeria berengeriana f. sp.piricola(L)treatment was slightly higher than Biocontrol bacteria (S) treatment. Then the enzyme activity of SOD treated by Biocontrol bacteria(S) gradually increased and reached high peak in 48 h, was 126.69 U/[g(FW)·min]which was 1.54 times of CK. The enzyme activity of SOD treated by Botryosphaeria berengeriana f. sp. piricola (L) as well as Botryosphaeria berengeriana f. sp.piricola and biocontrol bacteria (L+S)reached high peak in 24 h, were 122.10 U/[g (FW)·min]and 135. 32 U/[g (FW)·min]which were 1.48 and 1.65 times of CK respectively,but then decreased rapidly and dropped to the lowest point in 48h, were 56.62 U/[g(FW)·min]and 109.25 U/[g(FW)·min]respectively (Fig.2). On the whole, the effect of the enzyme activity of SOD treated by Biocontrol bacteria (S),Botryosphaeria berengeriana f. sp.piricola (L) as well as Botryosphaeria berengeriana f. sp. piricola and biocontrol bacteria (L+S) were all higher than CK, which indicated that both Botryosphaeria berengeriana f. sp.piricola and biocontrol bacteria could increase the excitation of SOD enzyme activity,but SOD enzyme activity had different response time to different treatment.

The results showed that the dynamic changes of POD enzyme activity of four treatments were basically the same,which increased at first and decreased subsequently(Fig.3). The enzyme activity of POD of 4 treatments had little difference before 12h and reached high peak in 24 h. Among them, the peak value of POD enzyme activity treated by Biocontrol bacteria(S) was highest, followed by Botryosphaeria berengeriana f. sp.piricola (L) treament as well as Botryosphaeria berengeriana f. sp.piricola and Biocontrol bacteria (L+S)treatment.The peak were 385.34 U/[g(FW)·min], 342. 50 U/[g (FW)·min]and 290. 00 U/[g (FW)·min]which were 1.83, 1.62 and 1.38 times of CK respectively. On the whole, the effect of POD enzyme activity treated by Biocontrol bacteria(S), Botryosphaeria berengeriana f. sp. piricola (L) as well as Botryosphaeria berengeriana f. sp.piricola and biocontrol bacteria (L+S)were all higher than CK,which indicated that both Botryosphaeria berengeriana f. sp. piricola and biocontrol bacteria could increase the excitation of POD enzyme activity. The enzyme activity of POD treated by Biocontrol bacteria(S)was the best,which means that Biocontrol bacteria might has stronger induction effect of POD enzyme activity.

The results showed that the dynamic changes of CAT enzyme activity of 4 treatments substantially increased at first and decreased subsequently, and the enzyme activity of CAT treated by biocontrol bacteria(S),Botryosphaeria berengeriana f. sp.piricola (L) as well as Botryosphaeria berengeriana f. sp. piricola and biocontrol bacteria (L +S) reached high peak in 6 h, were 133.33 U/[g(FW)·min],114.17 U/[g(FW)·min]and 113.35 U/[g (FW)·min]which were 1.33, 1.14 and 1.13 times of CK respectively(Fig.4). The enzyme activity of CAT treated by Biocontrol bacteria(S) declined rapidly and reached its lowest point in 24 h, then increased again.On the whole,the effect of CAT enzyme activity treated by Biocontrol bacteria(S), Botryosphaeria berengeriana f. sp. piricola (L) as well as Botryosphaeria berengeriana f. sp.piricola and biocontrol bacteria (L+S)were all higher than CK,which indicated that both Botryosphaeria berengeriana f. sp. piricola and biocontrol bacteria could increase the excitation of CAT enzyme activity. The influence of 3 treatments were relatively small compared to CK.

Dynamic changes of PPO activity

PPO is the primary enzyme for phenols material oxidation, which involved in phenols material oxidation in plants and generated Quinones,meanwhile involved in the synthesis of lignin, in order to kill and inhibit the propagation of pathogenic bacteriaand play a role in disease resistance[9].The results showed that biocontrol bacteria had little difference in CK; the enzyme activity of PPO of 4 treatments substantially increased at first and decreased subsequently, and the enzyme activity of PPO treated by Botryosphaeria berengeriana f. sp.piricola (L)reached high peak in 12 h,was U/[g (FW)·min]which was 1.76 times of CK; Botryosphaeria berengeriana f. sp. piricola and biocontrol bacteria (L +S) treatment reached high peak in 24 h, was 70.00 U/[g(FW)·min]which was 1.50 times of CK; CK and Biocontrol bacteria (S)reached high peak in 24h,and the difference was not obvious (Fig.5). On the whole, the effect of PPO enzyme activity treated by Biocontrol bacteria(S) was not obvious; Botryosphaeria berengeriana f. sp. piricola (L) treatment and Botryosphaeria berengeriana f.sp.piricola and biocontrol bacteria(L+S) treatment were higher than CK, which indicated that both Botryosphaeria berengeriana f. sp.piricola and biocontrol bacteria could increase the excitation of PPO enzyme activity.

Discussion

After infecting or inducing by pathogenic bacteria, the protective enzyme activity related to disease resistance increased, which was one of the important mechanism that induced?resistance. PPO, SOD, POD and CAT are important enzymes involved in plants that related to resisting pathogenic microorganism infection[10].In the determination of MDA, the enzyme activity of MDA treated by biocontrol bacteria(S) had little difference with CK, but the enzyme activity of MDA treated by Botryosphaeria berengeriana f. sp. piricola (L) as well as Botryosphaeria berengeriana f. sp.piricola and biocontrol bacteria (L+S)changed significantly, which indicated that Botryosphaeria berengeriana f.sp. piricola had more effect on MDA.From another perspective, biocontrol bacteria did not play a destructive role on plant tissues, but Botryosphaeria berengeriana f. sp. piricola damaged to cell structures and accumulated more reactive oxygen. In addition, the enzyme activity of MDA treated by Botryosphaeria berengeriana f. sp.piricola reached high peak in 48h,probably because Botryosphaeria berengeriana f. sp. piricola still expanded until the cell structures were completely destroyed. In the determination of SOD, the enzyme activity of SOD treated by Botryosphaeria berengeriana f. sp. piricola and biocontrol bacteria was highest, which indicated that the combined action of Botryosphaeria berengeriana f. sp.piricola and biocontrol bacteria had more effect on SOD. The enzyme activity of SOD treated by biocontrol bacteria reached high peak in 48 h, probably because biocontrol bacteria induced the activity of SOD slower, and whether the continue trend showed upward needed further study. In the determination of POD, the enzyme activity of POD treated by Botryosphaeria berengeriana f. sp.piricola and biocontrol bacteria (L+S)was lower than Botryosphaeria berengeriana f.sp. piricola(L) treatment and biocontrol bacteria (S) treatment in 24 h,probably because biocontrol bacteria inhibited the extension of Botryosphaeria berengeriana f.sp.piricola. In the determination of CAT, the conclusion of variation trend of CAT activity was inconsistent with the research results of Zhang linqing[10],whose results indicated that CAT activity of two garlic variety infected by Phanerochaete chrysosporium were lower than CK, and the specific reasons needed further study.

The research indicated that using biocontrol bacteria alone had different degree of effect on antioxidant enzyme system, probably because biocontrol bacteria could increase the excitation of autioxidant enzyme activity,in order to make pear fruits to start defense system to hedge against further damage in advance.Botryosphaeria berengeriana f. sp.piricola and biocontrol bacteria treatment generated a corresponding impact on antioxidant enzyme. But from the perspective of enzyme activity changes and the effect that use biocontrol bacteria and Botryosphaeria berengeriana f. sp. piricola alone, it does not fully indicated that biocontrol bacteria had inhibiting effect on Botryosphaeria berengeriana f. sp.piricola. Actually, the inhibition of biocontrol bacteria on plants pathogen was not a mechanism work alone, but the synergistic results of 2 or more than 2 kinds of antibacterial mechanism[11].

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