Resistance of Eggplant (Solanum melongena L.) to Verticillim wilt Correlates to Microbial Abundance and Soil Enzyme Activities

Aims: To determine the relationship between microbial abundance and enzyme activities of rhizosphere soil from different resistant eggplant cultivars, and resistance of eggplant to Verticillium wilt. Study design: The changes of microbial and enzymatic activities of the rhizosphere soil from different resistant eggplants after inoculation of Verticillium dahliae were analysed. Place and Duration of Study: The plants were grown in a plastic greenhouse of the Vegetable Crops Experimental Station, and the laboratory experiments were conducted at the Horticulture College, Shenyang Agricultural University from August to November, 2008. Methodology: 14 eggplant cultivars were selected and inoculated with Verticillium dahliae to screen their resistance against Verticillium wilt, and classified according the final disease index. The quantities of main cultivable microorganisms and some functional bacteria were investigated by the serial dilution method. Activities of oxidoreductase and hydrolase enzymes of rhizosphere soil were determined by spectrophotometry or colorimetric titrations. Results: The correlation analysis among resistance of eggplant to Verticillium wilt, microorganisms and enzyme activities showed that, the abundance of actinomyces, the ratios of bacteria to fungi and actinomyces to fungi, and the activities of catalase, polyphenol oxidase, protease and urease, were significantly positively related with the resistance.


INTRODUCTION
Verticillium wilt is a destructive disease in eggplant production, which is mainly caused by the infection of Verticillium dahliae through root surface to vascular system (Garibaldi et al, 2005;Wang et al., 2005). V. dahliae can survive in soil for more than 6 years, and infect many plant varieties, while chemical fungicides can't effect directly to the infected parthave no direct effects on infected plants, so the disease is hard to control all over the world (Pegg andBrady 2002；Ligoxigakis et al., 2002;Korolev et al., 2008;Berbegal et al., 2010). Therefore, it is necessary to consider the relationship among between plant, soil and pathogen synthetically, to manage this disease (Park et al, 1963;Han et al, 2006). Plant can affect the pathogen directly through root exudation, or modulate the soil conditions (such as the quantity of microorganisms and the activities of soil enzymes) indirectly (Bertin, 2003;Brusetti et al, 2004). Antagonistic rhizosphere bacteria, actinomycetes and fungi contribute to the induction of remarkable soil suppressiveness against V. dahliae (Marois et al, 1982;Berg et al, 1994;Tjamos et al, 2004). Meanwhile, beneficial rhizosphere microorganisms may increase plant growth and development indirectly, through the biocontrol of phytopathogens in the root zone (Weller, 1988;Chen, et al, 1990;Garbeva et al, 2004) and the enhanced availability of minerals (Davison, 1988;Murty and Ladha, 1988), as well as directly through the production of phytohormones (Patten and Glick, 1996). Soil enzymes are important for improving and maintaining soil fertility to ensure productivity, as they take part in organic matter decomposition and nutrient cycling (Verstraete and Voest, 1977;Sinsabaugh et al, 1991;Bohme et al, 2005;Mandal et al, 2007). Oxidoreductases (such as catalase polyphenol oxidase and peroxidase) act in the oxidation reduction in soil (Wang et al, 2011;Finkenbein et al, 2012), and hydrolases are involved in the N (e.g. protease, urease), C (e.g. cellulase, invertase), P (e.g. acid phosphatase) cycling and decomposition of cellulose, lignin, carbohydrate polymers and other biomacromolecules (Verstraete and Voest, 1977;Prieto et al, 2011;Wang et al, 2011). Several studies show that enzyme activities can be used as early indicators of changes in soil properties and microbial activity (Skujins, 1973;Ajwa et al, 1999;Kandeler et al, 2006;Makoi and Ndakidemi, 2008). All these regulatory actionsThe regulation of pathogens by the plants are is determined by the resistances of different varieties (Bertin et al, 2003;Wu et al, 2010). But the regulatory mechanisms of plant to soil microorganisms and enzyme activities, and their relationship with plant resistance, are not quite clear now. This paper has taken studied different resistant eggplant cultivars to define the relative soil parameters for disease resistance determination, to provide theoretical basis of soil environment management, and to define the direction for The disease was assessed on leaf symptoms by a wilt index from 0 to 4, according to Emmanouil and Wood (1981) and Xiao et al (1995). Health incidence, disease incidence and disease index were evaluated every 5 days since the first appearance of the typical wilt, using the following calculations:

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The eggplant was taken out from pot carefully, and shaken to remove the needless soil, only rhizosphere soil was retained for further determination. For each cultivar, the rhizosphere soil of 3 plants was taken and well mixed. Ten grams of rhizosphere soil was weighted, put into a 250 mL triangular flask containinged 100 mL of sterile distilled water, and shaken for 15 min (100rpm·min -1 ). Then after 5 min standing, 1 mL of supernatante were was decanteddiluted into 9 mL of sterile distilled water (10 -1 ), and then diluted the same way to different concentrations (from 10 -2 to 10 -9 ). The initial soil suspensions were oven dried to calculate the microbial abundance in each sampled soil. containing 5 mL of adapted liquid medium, per tube) containing Stephenson medium, modified (for nitrifying bacteria) and Иmшeheцkий's modified (for cellulose decomposing bacteria). Enumeration of nitrifying bacteria (using sulfanilic acid and α-naphthylamine as chromogenic agent) and cellulose decomposing bacteria (adding filter paper without cellulose to exam the exist of cellulose decomposing bacteria) grown in liquid medium was done with the method of most probable number (MPN, using the 3-tube MPN Table), after 15 days' incubation at 27 °C in dark. All these mediums were sterilized at 121 °C for 30 min. Each concentration was inoculated on three plates or tubes, which were three replications (Xvu and Zheng, 1986;Yan, 1988;Yin et al, 2009).

Determination of soil enzyme activities
The soils (from each cultivar) selected above were air-dried for 7 days, passed through a 1 mm screen, and mixed for the determination of enzymatic activities (Xuv and Zheng, 1986;Yan, 1988;Li et al, 2008;Gu et al, 2009;Zhao et al, 2012). The experiments were repeated three times.
The catalase activity was determined by KMnO 4 titration method. Two grams of soil samples wetted with 0.5 ml of methylbenzene, was were incubated for 0.5 h at 4°C. After that, 40 mL of distilled water and 5 mL of 0.3% H 2 O 2 (as substrate) were added, and oscillated shaken for 30 min (120 r·min -1 ), then added 5 mL of 3 mol·L -1 H 2 SO 4 were added immediately to terminate the reaction. The remaining H 2 O 2 was titrated by 0.01 mol·L -1 KMnO 4 , and the enzyme activity was defined by milliliters of 0.01 mol·L -1 KMnO 4 per gram of dry soil.
Samples without soil were used as backgroundcontrol.
The polyphenol oxidase and peroxidase activities were determined by means ofthe pyrogallol method, and expressed by the production of gallocatechin for 3 h. For determination of polyphenol oxidase activity, 10 mL of 1% pyrogallol and 2 mL of 0.5% H 2 O 2 were added to 1 g of soil in a 50 mL volumetric flask, and incubated at 30 °C for 3 h. The optical density of gallocatechin extracted by diethyl ether at 430 nm was measured to express the polyphenol oxidase activity. Samples without soil and without reaction substrate were used as backgroundcontrol. The activity of peroxidase was measured the same way without H 2 O 2 as substrate.
The protease activity was analyzed by the ninhydrin colorimetric method. For determination of protease activity, 2 g of soil samples was were wetted by 0.5 ml of methylbenzene and incubated for 24 h at 30 °C with 10 ml 1% of gelatin solution. After incubation, 0.5 ml of 0.1 mol·L −1 H 2 SO 4 and 3 ml of 20% Na 2 SO 4 were used to precipitate the proteins. The solutions of soil sample were centrifuged for 15 min (4000 rpm·min), then added 1mL of 2% ninhydrin solution was added. The mixture was extracted by boiled water for the color Comment [MF6]: Do you mean "sieve"? development, and diluted to 50 mL, then measured the optical density at 500 nm was measured. Samples without soil were used as backgroundcontrol. Finally, protease activity was expressed in terms of NH 2 -N per gram of dry soil for 24 h at 30 °C.
The cellulase activity was measured by the UV spectrophotometry method, and estimated through the production of glucose. For the determination of cellulase activity, 10 g of soil samples was were wetted with 2 mL of methylbenzene, then added 5 mL of acetate buffer (pH 5.5) and 5 mL of 1% carboxyl methyl cellulose (CMC) were added. The mixture was incubated at 37 °C for 72 h, then boiled to stop the reaction. Add 0.3 mg of KAl(SO 4 ) 2 ·12H 2 O was added to precipitate residual cellulose. The filtrate was diluted with distilled water to 50 mL, then 2 mL of diluent and 5 mL of 0.1% anthrone were mixed and boiled for 10 min to develop color.
The color intensity was measured at 551 nm. Blanks were incubated without substrate or soils.
The invertase activity was measured using sucrose as substrate, the soils (10g) was were mixed with 10 mL of 20% sucrose solution and 10 mL of phosphate buffer (pH 5.5) and incubated at 37 °C. After 24 hr, the mixture was diluted with distilled water to a final volume of 50 mL. Mixed 20 mL of diluent were mixed with 10 mL of Fehling reagent and 20 mL of distilled water, then added 3 mL of 33% KI and H 2 SO 4 (v/v 1:3) solutions were added. Used 0.1 mol·L -1 hyposulphite solution was used to titrate the mixture from bluebule to white, using starch as the indicator. Blanks were incubated without substrate or soils.
The urease activity was determined by the sodium phenate-sodium hypochlorite colorimetric method. As substrate, 10mL of 10% urea solution and 20 mL of citrate buffer (pH 6.7) were added to 10 g of soil samples before being incubated at 37°C for 24 h. After incubation, the soil samples were shaken for 30 min and filtrated. Poured 1 mL of filtrate was poured into a 50 mL volumetric flask, then added 4 mL of sodium phenate and 3 mL of sodium hypochlorite were added. After colourationcoloration, optical density at 578 nm was measured for the extractions within 60 min. Urease activity was expressed in terms of NH 4 + -N per 100 grams of dry soil for 24 h. Samples without soil and without reaction substrate were used as backgroundcontrol.
The acid phosphatase activity was measured by the disodium phenyl phosphate colorimetric method, and the activity unit was phenol per gram of dry soil in 24 h at 37 °C. A sample of 10 g of soils and 1.5 mL of methylbenzene were mixed, and added 10 mL of disodium phenyl phosphate (C 6 H 5 PO 4 Na 2 ·2H 2 O) and 10 mL of acetate buffer (pH 5.0, because the pH values of soil samples were less than 7.0) were added. After 24 h incubation at 37 °C, 1 mL of filtrate was transferred into a 100 mL volumetric flask, added 5 mL of borate buffer (pH 9.6) and 1 mL of Gibbs reagent were added to develop color. After diluting dilution to 50 mL volume,

Statistical analysis
The data were statistically analyzed with Excel software. Analysis of variance was performed using the Data Processing System software (DPS). The correlation coefficients and Standard Deviation (SD) were calculated by Statistics Package for Social Science software (SPSS).

Resistance to Verticillium wilt
According to the final disease index ( S-susceptible type. The same below.

Relationship between microflora abundance and resistance to Verticillium wilt
The abundance of microorganisms was higher in rhizosphere soils than in non-planting soil (CK). The quantities abundance of bacteria and actinomyces in rhizosphere soils of resistant cultivars were generally higher than in rhizosphere soil of other types, while the quantities abundance of fungi in soils of resistant types were lower than the average level (about 1.28x10 6 cfu·g -1 ) (Fig.1). The disease index of different resistant cultivars was significantly negatively correlated with the amount of actinomyces, but not correlated with the quantities of bacteria and fungi (Table 2). Meanwhile, the disease incidence was significantly negatively correlated with the abundance of bacteria and actinomyces, but not correlated with the abundance of fungi. The ratios of B/F (ratio of bBacteria to fFungi) and A/F (ratio of aActinomyces to fFungi) of resistant and moderate resistant cultivars were significantly higher than others. B/F and A/F were significantly negatively correlated with the disease index, with the correlation coefficients of -0.560 and -0.576.
The abundance of functional bacteria showed differences among cultivars, but were uncorrelated with disease incidence and disease index.

Relationship between soil enzyme activities and resistance to Verticillium wilt
As Table3 and Table 4 showed, the enzyme activities inof rhizosphere soil were higher thant in non-planting soil. Meanwhile, the enzyme activities mainly increased with the improvement of resistance. But the significant differences were only showed among few cultivars, which had greater difference in resistance levels, such as S. torvum (R), LY (MR) and XL (S). The enzyme activities of S. torvum and LY were extremely significant or significantly higher than XL.
The results of Table 5 showed that, activities of catalase, polyhenol oxidase, protease and urease were Note: Different small and capital letters mean significant differences from control at 0.05 and 0.01 levels respectively.
CK: non-planting soil. The unit of microbial abundance is 10 6 cfu·g -1 DM. Table 2 The   Note: Different small and capital letters mean significant differences from control at 0.05 and 0.01 levels respectively.
CK: non-planting soil. Numbers in parentheses are Standard Deviation (SD). The same below.  Note: **: Significant at 0.01 probability level; *: Significant at 0.05 probability level. The same below.  oxidase, peroxidase, protease, cellulaose, urease, and invertase, but not related to acid phosophatase activity. There was no significant correlation between acid phosophatase activity and the abundance of the rhizosphere microorganisms.

DISCUSSION
Rhizosphere is a microenvironment made up of root，soil and microorganisms (Lambers et al, 2009), could be more accurately defined as the volume of soil influenced by root activity (Hinsinger, 1998). Plants can affect the soil microorganisms and enzyme activities through root exudates (Landi, et al, 2006;Broeckling et al, 2008;Zhou B.L. et al, 2011;Gao et al, 2012). The effects vary with variety cultivars and are related to resistance of plant (Kong et al, 2008a(Kong et al, , 2008bBonkowski et al, 2009;Raaijmakers et al, 2009). Since rhizosphere microbial communities are strongly influenced by root exudates (Brant et al., 2006), it has been hypothesised that plants select for beneficial microbial communities in their rhizosphere .
Furthermore, plants may also play an important role in determining soil enzyme activities, as a mainly source of extracellular enzymes in soil (Martens et al, 1992;Gramss et al, 1999). But there is no unifying understanding of the correlation between resistance of plants, and soil microorganisms quantity abundance and enzyme activitiesy (Harper, 1950;Larkin, 1993;Li et al, 2007;Yin et al, 2009;Gu et al, 2009;. Study about the population of rhizosphere microorganisms of 6 cotton cultivars that have different resistance to V. dahliae has showed that, the diversity of populations of rhizosphere fungi and actinomyces are positively correlated with cotton resistance, but the diversity of rhizosphere bacteria population is not closely correlated with resistance (Li et al, 1998). Our experiment analyzed the changes of rhizosphere microbial quantity abundance and soil enzyme activity under infection of V. dahliae, which was completely different with from Li et al (1998), whose experiment was taken under natureal conditions without pathogens.
In our test, the soils was were sampled when disease incidences among cultivars varied. When the roots of susceptible cultivars were go browned and rotted, the root activities (including the excretive, absorptive and enzymatic activities et al) were decrease sharply, which leaded to the reduction of regulation and control to rhizosphere microorganisms and soil enzyme activities. Conversely, the resistant cultivars showed greater defense to prevent damages and maintain to root activity, so the regulation and control to rhizosphere soil remained at a high level. Especially, with the increase of B/F and A/F, and linked with the improvement of catalase, polyphenol oxidase, protease and urease activities, is correlated to the inhibition or the slowing down of the spread of V. dahliae was inhibited or slowed.
In this paper, the relationships between the resistance levels of different eggplant cultivars to Verticillium wilt,

Comment [MF18]: Precise what it means
the rhizosphere microbial population and the soil enzyme activities, were systematically analyzed. The ratios of B/F and A/F, the abundance of actinomyces and the activities of catalase, polyphenol oxidase, protease and urease in rhizosphere soil, showed positive correlation with the resistance level of eggplant to Verticillium wilt, so they could be useful indicators to assess soil health level and manage soil environment.
Further studies are needed on relevant physiological and biological process, to determine the possible composition of root exudates which might be involved in the soil management, and reveal the mechanism of root exudation. And with the progress in molecular biology techniques (Söederberg et al, 2004;Wei et al, 2006;Broeckling et al, 2008;), the isolation and identification of microorganisms should be more accurate to avoid the error caused by using traditional serial dilutionmicrobiological methods (only 1-4 % of the microorganisms in soil could be cultured in medium, Amann et al, 1995).

A A A ACKNOWLEDGMENT CKNOWLEDGMENT CKNOWLEDGMENT CKNOWLEDGMENT
The research was financially supported by the National Natural Science Foundation of China (31171950).