Intercropping with aerobic rice suppressed Fusarium wilt in watermelon

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Soil Biology & Biochemistry 40 (2008) 834–844 www.elsevier.com/locate/soilbio

Intercropping with aerobic rice suppressed Fusarium wilt in watermelon Lixuan Ren, Shiming Su, Xingming Yang, Yangchun Xu, Qiwei Huang, Qirong Shen College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, China Received 16 May 2007; received in revised form 31 October 2007; accepted 2 November 2007

Abstract Watermelon is susceptible to Fusarium wilt in successively mono-cropped soil. Pot experiments were carried out to investigate the effect of intercropping with aerobic rice on Fusarium wilt in watermelon. The tested soil was classified as a loam soil, previously planted with watermelon and collected from Hexian county, Anhui province, China. The results obtained are listed as follows: (1) 66.7% of watermelon plants were infected with wilt disease and 44.4% died on 40 days after transplanting in mono-cropped soil, but plants were much less susceptible to infection when intercropped with rice; (2) the density of Fusarium oxysporum f. sp. niveum decreased by 91% in soil from the intercropped watermelon rhizosphere when compared with that from the mono-crop 40 days after transplanting; (3) densities of bacteria and actinomycetes increased, but fungal density decreased in rhizosphere soil from the intercrops in comparison with the mono-crop control; (4) compared to the control, the germinated Fusarium spores were decreased by 41.0% in the treatment with addition of 1.5 ml rice root exudates. Adding 20 ml of root exudates decreased Fusarium spore production by 76.4%; and (5) the activities of defense enzymes in the leaves and roots of watermelons in the intercropped system were significantly lower than those in the monocropped system. It is suggested that intercropping with aerobic rice alleviated Fusarium wilt in watermelon, by restraining the spore production of Fusarium and by changing the microbial communities in rhizosphere soil through the production of rice root exudates. r 2007 Elsevier Ltd. All rights reserved. Keywords: Watermelon; Aerobically growing rice; Intercropping; Fusarium oxysporum f. sp. niveum; Microbial community; Rice root exudates

1. Introduction Watermelon (Citrullus lanatus (Trunb.) Matsum and Nakai) is an important fruit which is widely eaten around the world. However, this plant is usually subjected to attack by Fusarium wilt in continuously cropped soils, resulting in stunted growth and low production (Zhou and Everts, 2004). Chemical and biological methods have been used to alleviate and control Fusarium infection in watermelon. Chemical methods, such as spraying fungicide (Fravel et al., 2005) or using fumigant methyl bromide (Cebolla et al., 2000), are harmful to the environment (Song et al., 2004). Methods of biological suppression such as screening and planting resistant cultivars (Bai and Shaner, 1996), grafting (Miguel et al., 2004), using antagonists (Blanco et al., 2007; Fravel et al., 2005; Linderman, 1992) as well as compost amendment (BoulterCorresponding author. Tel.: +86 25 8439 6291/5212; fax: +86 25 8443 2420. E-mail address: [email protected] (Q. Shen).

0038-0717/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2007.11.003

Bitzer et al., 2006; Pavloua and Vakalounakis, 2005; Cotxarrera et al., 2002) have commonly been used, but most of them have not yielded consistent and reliable results. So far, very few studies have addressed alleviation of watermelon Fusarium wilt by intercropping cultivation. Rice is one of the most important cereal crops in the world. In recent years, reducing the consumption of water in rice production has received more attention due to water shortages around the world. Some researchers reported that rice yield in unsaturated soil by water-saving techniques was similar to that obtained by traditional waterlogging (paddy) methods (Qian et al., 2003). Furthermore, rice cultivation in unsaturated soils has made it possible to intercrop together with other plants. By rice intercropping with peanut, nitrogen fixation was improved and bi-directional nitrogen transfer occurred between peanut and rice in unsaturated soils (Shen and Chu, 2004; Chu et al., 2004). Thus, intercropping with rice grown under unsaturated conditions may be feasible in watermelon production. Microbial diversity is related to soil functions and ecosystem sustainability (Pankhurst et al., 1996; Kennedy

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and Smith, 1995). Brussaard et al. (2007) reported that the highest disease suppression was found in the plots with maximum soil microbial diversity. Similarly, the suppression of Fusarium wilt was related to microbial activity in soil (Borrero et al., 2004). Furthermore, intercropping system can alleviate plant disease by promoting soil microbial diversity. Tomato blight disease, for example, could be controlled in the tomato–marigold intercropping system (Go´mez-Rodrı´ guez et al., 2003). Apart from microbial diversity, other factors such as root exudates also played an important role in disease suppression. Gu and Mazzola (2003) reported that plant species or genotypes managed potential benefit to soil-borne diseases through root exudates. Furthermore, antagonistic potential in soils could be modified with changing cropping system (Gu and Mazzola, 2003; Vargas-Ayala et al., 2000). However, no reports have confirmed whether watermelon intercropped with rice can control the occurrence of Fusarium wilt disease in watermelon through changing microbial communities or releasing root exudates in soil. Malondialdehyde (MDA) content is considered as an index of membranous lipid peroxidation (Morsya et al., 2007), and generally increased under stressed conditions such as pathogenic infection (Heber et al., 1996). Apart from MDA content, stress-related enzymes such as catalases (CAT), peroxidases (POD), polyphenoloxides (PPO) and phenylalanine ammonia lyases (PAL) were induced and increased when the plants were subjected to pathogenic attack (Zhao et al., 2005; Rossum et al., 1997). In general, CAT and POD rapidly increased to protect membrane from H2O2 injury under stressed conditions (Zhou et al., 2003). PPO and PAL were induced to produce many secondary metabolites such as phenolic compounds, lignin etc. to protect membrane under stresses (Rossum et al., 1997). Therefore, CAT, POD, PPO and PAL activities are usually used to evaluate physiological and biochemical responses of plants to biotic and abiotic stresses (Gechev et al., 2003; Prasad, 1997). However, little information has been available regarding the changes of the above physiological parameters under watermelon–rice intercropping system to better understand the mechanism for alleviating watermelon wilt disease by intercropping with rice. The objectives of this study were to investigate: (1) watermelon wilt disease in response to intercropping with aerobic rice; (2) the changes in microbial communities of watermelon rhizosphere soil under intercropping with rice; (3) the effects of intercropping with rice on enzymatic activities related to plant resistance in watermelon; and (4) how rice root exudates affected conidial growth of Fusarium collected from watermelon plants with Fusarium wilt. 2. Materials and methods 2.1. Experimental design Three experiments were designed in this study. Experiment 1 was conducted to investigate how intercropping

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with rice affects Fusarium wilt infection in watermelon. The experiment was designed with two treatments: (1) watermelon (C. lanatus (Trunb.) Matsum and Nakai cv. Zaojia 84-24) monocropping and (2) watermelon intercropping with rice (Oryza sativa L. cv. 4007). In addition, control pots without plants were included. The soil used was classified as a loam soil and had been previously planted with watermelon and collected from Hexian county, Anhui province, China. The soil contained 23.48 g kg1 of organic matter, 0.90 g kg1 of total N, 34.54 mg kg1 of available P (Olsen), 141.3 mg kg1 of available K (NH4OAc), an electrolytic conductivity of 70 ms cm 1, and pH of 4.93 (1:1, soil:water ratio). After collection, the wet soil was airdried and passed through a 6 mm sieve to remove small stones and visible plant debris. An amount of 1.2 kg of soil was mixed with a compound fertilizer (N:P:K ¼ 14:16:15) at a rate of 0.96 g kg1 soil and put into plastic pots (12 cm  14 cm in diameter and height). All pots were arranged in a complete randomized block design with three replicates for each treatment. Rice seeds (O. sativa L. cv. 4007) were surface sterilized with 5% (v/v) H2O2 for 30 min, washed with deionized water and then directly sown into the potted soils. Fifteen days after emergence, rice seedlings were thinned and watermelon seedlings with three leaves were transplanted into the pots. The intercropping treatment, each pot contained two rice seedlings as one cluster and two watermelon seedlings. The monocropping treatment, three watermelon seedlings were transplanted into the pots. All plants were distributed as triangle in each pot. The seedlings in the pots were kept 6 cm away from each other. The seedlings were grown under controlled conditions (30/ 18 1C) for 40 days. The pots were daily irrigated with deionized water to keep soil water content at 80% of water holding capacity (WHC). At harvest, plants were collected and separated into shoots and roots. Leaf samples were immediately frozen with liquid nitrogen and kept at 70 1C to measure MDA content and the activities of CAT, POD, PPO and PAL. The residual part of the shoot was weighed to get fresh weight. Watermelon roots were taken from the pots and gently shaken by hand to get the soil adhered on the roots, hereafter referred to as rhizosphere soil. The more distant soil from root was regarded as bulk soil. The roots were washed for 10 min with tap water to remove soil particles and other impurities, and then with deionized water. The collected root samples were immediately frozen with liquid nitrogen and stored at 70 1C until analysis. One portion of each soil sample (i.e. rhizosphere soil, bulk soil and control soil) was kept at 18 1C to analyze microbial densities including Fusarium, bacteria, actinomyces and fungi using a plate dilution method. Another portion of each soil sample was stored at 70 1C for PLFA analysis. Experiment 2 was performed to investigate the effects of intercropping with rice on the microbial community in the rhizosphere soil with watermelons. This experiment was designed with two treatments: (1) monocropping with

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watermelon and (2) watermelon intercropping with rice. The soil and fertilizer were same as those in experiment 1. Five kilograms of basally fertilized soil was filled into experimental pots (20 cm  24 cm in diameter and high). Both plants were grown at same method with experiment 1. Two clusters of four rice seedlings were maintained at the diametric direction and grown in each pot for intercropping system. Watermelon seedlings with three leaves were transplanted at four seedlings per pot in the monocropping treatment and two seedlings in the intercropping treatment, in which two watermelon seedlings were oriented at the vertical diametric direction with the two clusters of rice seedlings. The seedlings in the pots were kept 8–10 cm away from each other. All pots were arranged in a completely randomized block design with four replicates of each treatment. Rhizosphere soil from the watermelon was sampled every 10 days after transplanting. Soil samples were taken directly from the pot at 2 cm around the watermelon plant using a mini-auger (2 cm diameter). Each soil sample (10 g) was a mixed sample taken from two spots in each pot. Prior to sampling, surface soil was gently removed by hands until the roots could be seen. After that, the mini-auger was gently inserted into the soil to avoid injuring the thicker roots of the watermelon plants. After sampling, the adjacent soil was used to back-fill the holes. The soil samples were kept under the same conditions as described in experiment 1 to measure the densities of Fusarium, bacteria, actinomycetes and fungi using a plate dilution method. Experiment 3 was conducted to investigate the effect of rice root exudates on conidial growth of Fusarium oxysporum f. sp. niveum. The pathogens were isolated from infected watermelon plants. The separated colonies were inoculated on Bilay’s media (Hartman et al., 2004), and incubated at 28 1C in a mechanical shaker at 120 min1 for 4 days to get spore supernatants. The supernatants were filtered through cheesecloth to remove mycelial fragments. The filtrate was centrifuged (6000  g for 15 min, 4 1C) to get the spores. The collected spores were washed with deionized water and centrifuged for three times. Finally, the spores were re-suspended in sterile deionized water. 0.1 ml of spore suspension was incubated on the PDA substrate. The spore number was counted 3 days after incubation and then the suspensions were frozen at 20 1C. Rice (O. sativa L. cv. 4007) seedlings were grown in a growth tank (40 cm  30 cm  20 cm) filled with rich mineral growth medium by International Rice Research Institution (IRRI) in a phytotron at 30/22 1C day/night (Watanabe et al., 1977). Root exudates were collected after seedlings had been growing in the growth tank for 4 weeks. Rice seedlings were gently taken out of the nutrient solution. The roots were washed with running tap water and deionized water. The seedling roots were immersed in plastic cups containing 300 ml of deionized water. Each cup contained six seedlings. A black plastic lid with a hole was covered on the cup to avoid contamination and light. Four hours later, root exudates were collected from the cup. The

collected root exudates were concentrated to almost dry using a rotary evaporator at 40 1C. The residue was dissolved by 10 ml of deionized water and then filtered twice by millipore filters (0.45 and 0.22 mm) and frozen at 20 1C. 2.2. Analysis 2.2.1. Plate dilution method The densities of culturable bacteria, actinomycetes and fungi were estimated using a standard dilution-plating procedure. The Fusarium, bacteria, actinomycetes and fungi were incubated with Komada’s (1975) Fusarium selective substrate, beef broth peptone substrate, Gause No. 1 substrate and Martin substrate, respectively. Four plates were measured for each parameter of each soil. 2.2.2. PLFA analysis Lipids were extracted by a modified Bligh–Dyer method (Bossio and Scow, 1995; Bligh and Dyer, 1959). Briefly, 4.5 ml of a one-phase mixture of chloroform–methanol–citrate buffer solution (1:2:0.8, v/v/v) was added to 3 g of frozen and dried soil. After centrifugation (repeated twice), a mixture of citric acid–chloroform (1:1, v/v) of 6.2 ml was added into the supernatants. Then, the mixed solution was swirled for 30 s and centrifuged at 4000  g for 10 min. The extracted lipids, present in the chloroform phase were dried under nitrogen gas. The samples were then fractionated into neutral, intermediate, and polar lipids by elution with 5 ml of chloroform, 5 ml of acetone, and 5 ml of methanol, respectively, using silicon acid bonded phase columns previously conditioned with 0.3 ml of chloroform. The methanol phase was collected and lipid fatty acid of 14:0 was added as a standard. The methanol phase with sample and standard was dried under nitrogen. The samples were then subjected to alkaline methanolysis using methanolic KOH 0.2 M, which trans-esterified fatty acids derived from neutral and phospholipids into free fatty acid methyl esters. The methylated compounds were analyzed by gas chromatography (AgilentGC-6890N). Different fatty acids were considered to represent different microorganisms. For example, the Gram-positive bacteria were represented by the sum of: i14:0, i15:0, a15:0, i16:0, a16:0, i17:0 and a17:0 (Kourtev et al., 2003; Zelles et al., 1994). Sum of the following fatty acids was considered to represent Gramnegative bacteria: 14:0, 16:1o7c, 16:1o9, 17:0, cy17:0, 18:1o5, 18:1o7c and cy19:0 (Kourtev et al., 2003; Jain et al., 1997). As markers for actinomycetes 10Me16:0 and 10Me17:0 were used (Macnaughton and Donnell, 1994). Fungi were represented by 18:2o6,9 (Frostegaa˚rd and Ba˚a˚th, 1996). The ratio of the sum of bacteria to fungi was represented by bacterial PLFAs /18:2o6,9. Fatty acid 16:1o5c was considered as an indicator for arbuscular mycorrhizal fungi (Kourtev et al., 2003; Drijber et al., 2000; Olsson and Alstro¨, 2000), but also occurred in bacterial species (Drijber et al., 2000; Olsson and Alstro¨, 2000). In this study, 16:1o5c was regarded as a single and

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represented AM fungi. PLFA content was denoted by mol% PLFA. Each measurement was replicated three times. 2.2.3. MDA content and enzymatic activities MDA content was analyzed by the Thiobarbiturin acid (TBA) coloration method (Heath and Packer, 1968). A 0.5 g of leaf or root samples was homogenized in 0.3% TBA with 10% trichloroacetic acid (TCA) at 4 1C. MDA content was calculated from the difference in absorbance between 532 and 600 nm using the extinction coefficient of 155 mmol1 cm1 and expressed as mmol MDA g1 fresh weight. For determining the activities of CAT, POD and PPO, 0.5 g of leaf or root samples was homogenized in 50 mM Na-phosphate buffer (pH 6.5) containing 2% (w/v) polyvinypyrrolidone (PVP) and 1 M NaCl. The homogenate was centrifuged (18,000g, 4 1C) to collect supernatant for enzyme assays. CAT activity was determined according to the method by Kraus and Fletcher (1994) with modification. One unit of CAT was defined as the decrease in absorbance of 0.01 U min1 at 240 nm. POD activity was assayed as described by Hammerschmidt and Kuc (1982) with modification. The assay mixture consisted of guaiacol and H2O2 in sodium phosphate buffer. The reaction product was measured with spectrophotometer at 470 nm. One unit (U) of POD was defined as the increase in absorbance of 0.01 U min1. PPO activity was measured by incubating enzymatic extract in sodium phosphate containing catechol for 1 min and monitoring the change of absorbance at 398 nm by spectrophotometer (Wang et al., 2004). For PAL activity, 0.5 g of samples was extracted in 0.1 M borate buffer, pH 8.8 with 14 mM 2-mercaptoethanol. The homogenate was centrifuged to collect supernatant as enzyme extract. PAL activity was determined according to the production of trans-cinnamic acid from L-phenylalanine spectrophotometrically at 290 nm (Mozzetti et al., 1995). Each measurement was replicated three times. 2.2.4. Spore germination, mycelial growth and spore reproduction For spore germination, sterilized PDA (potato dextrose agar) substrate was poured into 9 cm plates. After the substrate solidified, the solution of root exudates (0.00, 0.15, 0.45, 0.75, 1.05, 1.35 and 1.50 ml plus sterilized water to 1.5 ml) was pipetted into PDA plates. The treatment without root exudates was included as control. 0.1 ml of spore supernatants (2  103 cfu/ml) was added to the plates. The plates were gently turned by hands to thoroughly mix root exudates with spore supernatants and incubated at 28 1C. The colonies were counted to indicate the germinated spores after 3 days of incubation. For mycelial growth, sterilized PDA substrate was mixed with root exudates (0, 0.15, 0.45, 0.75, 1.50, 3.00 and 6.00 ml) to make 15 ml of total volume. The components of

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PDA substrate were consistent in different treatments. The complex substrate was poured into a U-type tube. After the substrate solidified, 9 mm diameter colonies of Fusarium were inoculated on the substrate surface. The colonies were incubated at 28 1C and mycelial length was measured every day for 12 days after inoculation. For spore reproduction, sterilized Bilay’s substrate was added into rice root exudates (0, 0.5, 1.5, 5.0, 10.0, 15.0 and 20.0 ml) to make 50 ml of total volume. The components of Bilay’s substrate were consistent in different treatments. 0.1 ml of Fusarium spore suspension (2  103 cfu/ml) was inoculated into Bilay’s substrate with different amounts of rice root exudates, and cultured at 28 1C in a mechanical shaker at 120 min1 for 3 days. The spore supernatant was filtered and re-suspended, and then the spore number was recorded. Each treatment was replicated four times. 2.3. Statistical analyses All data were expressed as mean7standard error and subjected to ANOVA. The canonical score plots on the PLFA was analyzed by discriminant analysis using the SPSS software (version 11.5). The other data were analyzed by Tukey’s or t-tests method (Po0.05) using the SAS software (version 8.2). 3. Results 3.1. Plant growth and Fusarium wilt in watermelon as affected by intercropping with aerobically growing rice In experiment 1, intercropping with rice improved growth and alleviated wilt disease in watermelon. Transplanted watermelon plants grew at a slower rate under monocropping than under intercropping with rice (Table 1). 40 days after transplanting, both fresh weight and plant height of watermelons in monocropping treatment were lower than those in intercropping treatment. Watermelons in monocropping system began to show Fusarium wilt on 25 days after transplanting. On 40 days after transplanting, some of the watermelons were infected with wilt disease and died in monocropping system. However, they grew normally in intercropping system. 3.2. Microbial densities in the rhizosphere soil of watermelons as affected by intercropping with rice In experiment 1, when compared with the control, Fusarium density in the rhizosphere soil increased in monocropping treatment, but decreased in intercropping treatment 40 days after transplanting (Fig. 1). The density of Fusarium in rhizosphere soil with mono-cropped watermelon was 1867 g1 soil, while only 167 g1 soil in rhizosphere soil with intercropped watermelon. The Fusarium density in rhizosphere soil of intercropped watermelon declined by 91%, compared with that in the

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Table 1 The effects of intercropping with rice on the growth and Fusarium wilt infection of watermelons Treatments

FW (g)

Plant height (cm)

Rate of Fusarium wilt (%)

Mortality rate (%)

Intercropping watermelon Monocropping watermelon

10.64a 3.72b

35.2a 19.5b

0b 66.7a

0b 44.4a

Fusarium density (× 102 g-1)

Note: FW—fresh weight. The data with the same letters in a column were not significantly different by independent samples T test (Pp0.05).

25 a

20 15 b

b

10 5

c c

0 CK

MR

MB Treatments

IR

IB

Fig. 1. The effects of intercropping with rice on Fusarium density in the rhizosphere and bulk soil of watermelon. In this figure: CK—the tested soil without plants; MR—rhizosphere soil from monocropping watermelon; MB—bulk soil from monocropping watermelon; IR—rhizosphere soil from intercropping watermelon; and IB—bulk soil from intercropping watermelon. Vertical bars indicate the standard error of the averages. The columns with the same letter were not significantly different by Tukey test method at Pp0.05.

monocropped. No significant difference in Fusarium density was detected in bulk soil under both cropping treatments. Differences in Fusarium density also existed between bulk soil and rhizosphere soil, regardless of cropping systems. Fusarium density in bulk soil was lower than rhizosphere soil in monocropping system, but the reverse was true under intercropping. Microbial diversity in soil is shown in Table 2 (experiment 1). The densities of bacteria, actinomycetes and total microbes in rhizosphere soil were markedly higher under intercropping, compared with monocropping. In contrast, fungal density in rhizosphere soil was higher under monocropping than intercropping systems. In general, microbial densities in rhizosphere soil were higher than those in the bulk soil. The diversity of microbial community structure in soil was examined by PLFA. 38 PLFAs were detected and discriminant analysis was used to pinpoint microbial community structure in different soils. The canonical score plot showed that soil microbial community structures were distinctly clustered into five groups (Fig. 2, experiment 1). The first two canonical functions (Wilk’s lambda o0.0001, Po0.0001) explained 98% of the variation in the data set. Therefore, the predefined grouping used in the discriminant analysis was justified. These results suggested that microbial community structures were different among different soils, including rhizosphere soil of mono-cropped water-

melon, bulk soil of mono-cropped watermelon, rhizosphere soil of intercropped watermelon, bulk soil of intercropped watermelon and control soil. Thirty dominant PLFAs were selected to interpret microbial community structure. The characteristic fatty acids were dominant in the tested soil samples, such as Gram-negative bacteria (14:0, 16:1o7c, 16:1o9, 17:0, cy17:0, 18:1o5, 18:1o7c and cy19:0), Gram-positive bacteria (i14:0, i15:0, a15:0, i16:0, a16:0, i17:0 and a17:0), actinomycetes (10Me16:0 and 10Me17:0), fungi (18:2o6,9) and arbuscular mycorrhiza (16:1o5c). Table 3 (experiment 1) showed that microbial community structure in intercropped rhizosphere soil was different from mono-cropped rhizosphere soil. The PLFAs of Gram-positive bacteria, actinomycetes and 16:1o5c in rhizosphere soil under intercropping were higher than monocropping. Furthermore, the ratio of bacteria (sum of Gram-negative and Gram-positive) to fungi was high but the ratio of Gramnegative to Gram-positive was low in rhizosphere soil under intercropping, in contrast to monocropping. In addition, regardless of intercropping or monocropping, microbial community structures in rhizosphere soils were different from other soils (bulk soils and control soil). The actinomycetes in rhizosphere soils were higher, while fungi were lower than other soils. Both ratios of bacteria to fungi and actinomycetes to fungi in rhizosphere soils were higher than other soils. The fatty acid of 16:1o5c was the highest in rhizosphere soil of intercropping treatment among all soils. These results suggested that bacteria, especially Gram-positive bacteria and actinomycetes in rhizosphere soil of intercropped watermelon increased, compared to other soils. In experiment 2, no difference in Fusarium density was observed in rhizosphere soils of watermelon between monocropping and intercropping within 20 days after transplanting (Fig. 3). After that, Fusarium density continued to increase under monocropping, but decreased under intercropping. Thirty days after transplanting, Fusarium density was significantly lower in intercropping treatment than monocropping treatment. From 40 days after watermelon transplanting, bacterial density and total microbial densities became higher under intercropping, compared with monocropping system. Actinomycetes also showed a similar trend to bacteria at 50 days after transplanting. By contrast, fungal density dramatically decreased under intercropping in comparison with monocropping from 40 days after transplanting (Fig. 4, experiment 2).

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Table 2 The effects of intercropping with rice on the microorganism density in rhizosphere and bulk soil of watermelon Treatments

Bacteria (  106)

Actinomycetes (  106)

Total Fungi (  103)

Total microorganisms (  107)

CK MR MB IR IB

48.67712.01c 88.33732.02b 15.0076.08c 204.33721.73a 84.0076.00b

2.3370.47c 43.0079.90a 11.0071.63bc 58.3379.98a 26.0077.79b

11.0070.82b 11.6771.70ab 9.3371.25b 9.0071.41b 15.0072.16a

5.1071.15c 13.1372.93b 2.6070.66c 26.2771.10a 11.0071.32b

Note: CK—no plant; MR—rhizosphere soil from monocropping watermelon; MB—bulk soil from monocropping watermelon; IR—rhizosphere soil from intercropping watermelon; and IB—bulk soil from intercropping watermelon. The data with the same letters in a column were not significantly different by Tukey test method (Pp0.05).

15

4

1 2

10

2

3 4

Function 2

5

5 Group Centroid

0 -5 5 1

-10 -15 -25

0

25 Function 1

50

75

Fig. 2. Canonical score plots on the PLFA with discriminant functions. In this figure: 1—the tested soil without plants; 2—rhizosphere soil from intercropping watermelon; 3—bulk soil from intercropping watermelon; 4—rhizosphere soil from monocropping watermelon; 5—bulk soil from monocropping watermelon; and ’—group centroid.

3.3. Changes of MDA content and enzymatic activities in watermelons as affected by intercropping with rice Watermelon–rice intercropping decreased MDA contents by 66.3% in leaves and 51.5% in roots of watermelon, compared to monocropping. Similarly, intercropping with rice also decreased the activities of CAT, POD, and PPO in leaves and roots in watermelons, when compared with monocropping, with 44.8% and 89.0% decrease in leaves and roots for CAT, 74.1% and 55.7% for POD, 59.1% and 65.5% for PPO, respectively. Furthermore, PAL activity was decreased by 92.0% in roots with intercropping, in contrast to monocropping, but no significant difference in leaves between the two cropping treatments could be detected (Table 4, experiment 1). 3.4. Rice root exudates effects on Fusarium Spore germination and reproduction of Fusarium were significantly inhibited by rice root exudates. These inhibi-

tory effects were enhanced with increasing the concentration of root exudates. The inhibition of spore germination occurred with adding 0.45 ml of root exudates. When 1.5 ml root exudates were added, the germinated spore number was 111 g1 soil, which was declined by 41.0% compared to the control with 188 g1 soil (Fig. 5A). Spore reproduction of Fusarium was significantly depressed by adding rice root exudates. Compared to the control with 334  105, the spore number was 172  105 and decreased by 48.5% with adding 0.5 ml of root exudates. The spore numbers were significantly decreased with increasing addition of root exudates. Compared with the control, adding 20 ml of root exudates reduced the spore number by 76.4% and showed 79  105 (Fig. 5B). In addition, adding root exudates had no influence on mycelial length of Fusarium.

4. Discussion 4.1. Watermelon–rice intercropping controlled watermelon Fusarium wilt This experiment showed that intercropping with aerobically growing rice controlled Fusarium wilt disease in watermelon. Intercropping with aerobically growing rice increased fresh weight and plant height of watermelon. No watermelon seedlings were infected with Fusarium wilt in intercropping system. The reasons may be related to that intercropping with aerobically growing rice improved soil environment in watermelon, especially microbial diversity in soil (Fig. 1 and Table 2), and induced physiological changes to protect the watermelon from injury (Table 4). This result was similar with the effect of tomato–marigold intercropping system on suppression tomato blight disease (Go´mez-Rodrı´ guez et al., 2003). Garlic intercropped with Brassica could also alleviate white rot of garlic (Zewde et al., 2007). Rao and Mathuva (2000) reported that pigeonpea–maize intercropping system produced higher maize yield and income than continuous sole maize. Accordingly, watermelon production and income may also be improved by intercropping with rice decreasing its disease.

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Table 3 Presence and proportion of major PLFAs in the soils of different treatments (mol% ) Treatments

CK

MR

MB

IR

IB

G() G(+) AC Fungi 16:1w5c G()+G(+) G()/G(+) BA/Fungi AC/Fungi

35.2373.19a 15.1470.81ab 5.9770.49b 3.0670.14a 1.2470.06cd 50.3772.55a 2.3470.32ab 16.4470.09bc 1.9670.24b

29.4370.28c 14.0271.19b 6.4970.17ab 2.5870.36b 1.2970.04bc 43.4471.34b 2.1170.17ab 17.1070.53b 2.5570.41a

29.3971.22c 15.1771.21ab 6.4270.19ab 3.3370.23a 1.4470.15b 44.5672.33b 1.9470.10bc 13.4471.47d 1.9370.13b

30.4571.20bc 16.4770.40a 6.7170.08a 2.4970.02b 2.3470.13a 46.9271.60ab 1.8570.03c 18.8270.80a 2.6970.03a

34.0073.52ab 13.5770.46b 6.0670.54b 3.1270.18a 1.0970.03d 47.5773.09ab 2.5170.34a 15.2370.13cd 1.9570.29b

Note: CK—no plant; MR—rhizosphere soil from monocropping watermelon; MB—bulk soil from monocropping watermelon; IR—rhizosphere soil from intercropping watermelon; IB—bulk soil from intercropping watermelon; G ()—Gram-negative; G (+)—Gram-positive; AC—actinomycetes; G ()+G (+)—the sum of G () and G (+); and BA—bacterium—the sum of G ()+G (+). The data with the same letters in a row were not significantly different by Tukey test method (Pp0.05).

1750 intercropping

Fusarium density (g-1)

1500

monocropping

a

1250 1000

a

750 500

b b

250 0 0

10 20 30 Days after transplanting

40

Fig. 3. Dynamic variation of Fusarium density in near rhizosphere soil of watermelon in intercropping and monocropping systems. Vertical bars indicate the standard error of the averages. The simultaneous sampling data with the same letter were not significantly different by independent samples T test at Pp0.05.

4.2. Watermelon–rice intercropping suppressed pathogen and impacted microbial communities in rhizosphere of watermelon It is readily acknowledged that pathogen suppression in soil can alleviate plant disease. Secondarily, the soil defensive capability increased as the abundance and diversity of the microbial community became greater (Janvier et al., 2007; Sturz and Christie, 2003). Brussaard et al. (2007) also reported that the highest disease suppression was found in plots with greatest soil microbial diversity. Moreover, Dommergues (1978) classified rhizosphere microorganisms into having beneficial, harmful, or no effect on the plant. Many species of bacteria and actinomycetes can cause plant disease but more damage are caused by fungi (Brussaard et al., 2007). Some bacteria have the potential for pathogen suppression (Boulter et al., 2002). In successively mono-cropped soil, specific microbial communities were formed due to the accumulation of specific exudates (Brussaard et al., 2007; Janvier et al.,

2007). In this study, watermelon–rice intercropping markedly suppressed Fusarium pathogen and changed soil microbial communities: (1) Intercropping with rice decreased Fusarium density in rhizosphere soil of watermelon, in contrast to monocropping; (2) with plate diluting method, intercropping with rice increased bacteria and total microorganisms but decreased fungi in rhizosphere soil of watermelon, compared to monocropping; and (3) with PLFA method, intercropping with rice increased Gram-positive and actinomycetes in rhizosphere soil of watermelon, compared to monocropping. Therefore, besides suppressed Fusarium density, the changed microbial communities by intercropping with rice such as improved microbial biodiversity and increased Gram-positive may be the other reason for alleviating the occurrence of Fusarium wilt in watermelon under intercropping with rice. Microbial communities are likely to alleviate wilt disease by some antagonist suppressed pathogen or by some parasitic colonization reduced ability of pathogen infection (Simon and Sivasithamparam, 1989; Cook and Baker, 1983; Schneider, 1982). Dynamic variations of Fusarium density and microbial densities showed that, in intercropping system, Fusarium density decreased from 30 days after transplanting while the significant difference of other microbial densities between intercropping and monocropping produced from 40 days after transplanting. Therefore, the antagonistic microorganism in watermelon rhizosphere soil was not the inducement of Fusarium suppression. Furthermore, Larkin et al. (1993a, b) demonstrated that soil suppression to Fusarium wilt are associated with microflora population caused by different plant, but the general populations of microorganism were not involved in Fusarium suppression. Therefore, microbial communities just reduced the Fusarium infection rather than suppressed Fusarium directly. In addition, the fatty acid 16:1o5c mol% is higher in rhizosphere soil of intercropping watermelon than that in all other soils (Table 3). The fatty acid 16:1o5c is considered as an indicator for arbuscular mycorrhizal fungi, although it has also been found in bacterial species (Drijber et al., 2000; Olsson and Alstro¨,

ARTICLE IN PRESS L. Ren et al. / Soil Biology & Biochemistry 40 (2008) 834–844 intercrop

monoculture 18

16

16

14

a

14 a

12 10

10

a

8

8

a

6

6

b 4

b

b

3

2

4 2

0

0 0

10

20

30

40

0

50

10

20

30

40

50 3.0

12 a 10

Fungi density (×103 g-1)

12

a

actinomycetes density (×106 g-1)

monoculture

18

a

a a

8 6

2.0

a a

2.5

a

a

a

1.5

a

a

a

4

b

b

1.0

b

a

0.5

2

Microoganism density (×107 g-1)

Bacteria density (×106 g-1)

intercrop

841

0.0

0 0

10

20 30 Days after transplanting

40

0

50

10

20 30 Days after transplanting

40

50

Fig. 4. Dynamic variation of microorganism density near rhizosphere of watermelon in different treatments: (A) bacteria; (B) actinomycetes; (C) fungi; and (D) total microorganism. Vertical bars indicate the standard error of the averages. The simultaneous sampling data with the same letter were not significantly different by independent samples T test at Pp0.05. Table 4 The effects of intercropping with rice on MDA content and defensive enzyme activity in roots and leaves of watermelon Treatments

MDA (mmol g1 FW)

CAT (U min1 mg1 FW)

POD (U min1 mg1 FW)

PPO (U min1 mg1 FW)

PAL (U h1 mg1 FW)

ML IL MR IR

17.4474.61a 5.8770.59b 2.1670.03c 1.0570.57d

7.9370.31a 4.3870.57b 3.4770.10c 0.3870.01d

3.2170.22c 0.8370.10d 29.6571.68a 13.1370.48b

3.3770.81a 1.3870.30c 2.2170.39b 0.7670.05d

0.4770.09b 0.4670.02b 1.8370.57a 0.1570.06c

Note: ML—monocropping watermelon leaves; IL—intercropping watermelon leaves; MR—monocropping watermelon roots; and IR—intercropping watermelon roots. The data with the same letters in a column were not significantly different by Tukey test method (Pp0.05).

2000). Both rice and watermelon can form arbuscular mycorrhizae. Furthermore, arbuscular mycorrhizae take important function in disease resistance of plant (Khaosaad et al., 2007; Caron et al., 1986). This result implied that more attention should be paid to arbuscular mycorrhizae fungi in watermelon–rice intercropping system in future research. 4.3. Physiological changes in watermelon–rice intercropping system MDA content in plants is generally used for evaluating the injury degree under stressed conditions (Heber et al.,

1996). In this study, MDA content in watermelon kept low values under intercropping with rice, in contrast to monocropping (Table 4). The activities of CAT, POD, PPO and PAL are usually used to evaluate physiological and biochemical responses of plants to biotic and abiotic stresses (Gechev et al., 2003; Prasad, 1997). The activities of the above enzymes in plant were increased under biotic stresses as pathogenic fungi (Sudhakar et al., 2007; Melo et al., 2006; Jang et al., 2004; Li and Burritt, 2003). In this study, intercropping with rice decreased MDA content, the activities of CAT, POD and PPO in leaves and roots, as well as PAL activity in roots, in comparison with monocropping, implying that intercropping increased the

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842

Number of spores

250 200

a

a b c

150

c d

d

1.35

1.5

d

d

15

20

100 50 0 ck

0.15

0.45 0.75 1.05 Amount added (ml)

Number of spores (×105)

500 400

a

300 b

200

bc

bcd cd

100 0 CK

0.5

1.5

5

10

Amount added (ml)

Fig. 5. Effects of rice root exudates on spore germination and spore reproduction of Fusarium: (A) spore germination and (B) spore reproduction. Vertical bars indicate the standard error of the averages. The columns with the same letter were not significantly different by Tukey test method at Pp0.05.

stability of biological membranes to prevent watermelon from pathogenic infection.

needed to identify the functional components in rice root exudates for restraining Fusarium. In conclusion, intercropping with rice greatly improved watermelon growth in a continuously monocropped soil, significantly alleviated occurrence of Fusarium infection in watermelon. It is suggested that intercropping with rice alleviated Fusarium wilt in watermelon by inhibiting pathogen spore production and by changing microbial communities in rhizosphere soil through influencing root exudates. However, as this study was performed all in pot and laboratory experiments, further research is needed to investigate the feasibility of intercropping with rice to control Fusarium wilt in watermelons under field conditions. The following two aspects should be considered in performing field experiments. One aspect is the suitable horizontal distance between watermelon and rice to guarantee that the rice root has effects on Fusarium spore around watermelon root through root exudation, and that both crops should have enough space to normally grow and develop with optimal outcome. Another is the temporal interval between germinating rice and watermelon. The present pot experiments indicated that 6–10 cm horizontal distance between watermelon and rice, and oneweek interval for germinating rice and watermelon are suitable for this intercropping system, but the feasibilities need further investigations in the field. Acknowledgments We gratefully acknowledge the 973 programs, basic research on fertilizer saving and efficiency improvement for sustainable utilization of farmland, from China Science and Technology Ministry. We also thank Dr. T. Miller from UK for carefully checking the manuscript. References

4.4. Rice root exudates suppressed Fusarium Allelochemicals are defined as compounds with low molecular weight excreted from plants in the process of secondary metabolism and accumulated in plants, soils and other organisms (Rice, 1979). These compounds could inhibit or promote other plant species by producing allelopathic substances in root exudates (Singh et al., 2007; Yu et al., 2000; Yu and Matsui, 1993). Allelochemicals showed autotoxicity in continuously monocropped soils (Hao et al., 2006), but could alleviate autotoxicity (as diseases) through intercropping with other crops (Go´mezRodrı´ guez et al., 2003). Go´mez-Rodrı´ guez et al. (2003) reported that intercropping with marigold decreased the occurrence of blight disease in tomato through allelopathic effect of marigold on A. solani conidia germination. Similarly, the present results showed that rice root exudates suppressed pathogenic Fusarium growth, such as restrained spore germination and spore production (Fig. 5), which accounted for the alleviation of Fusarium in watermelon by intercropping with rice. Certainly, further research is

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