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Companion cropping with wheat increases resistance to Fusarium wilt in watermelon and the roles of root exudates in watermelon root growth
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Contents lists available at ScienceDirect
Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp
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Companion cropping with wheat increases resistance to Fusarium wilt in watermelon and the roles of root exudates in watermelon root growth
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Weihui Xu a, b, c, d, 1, Zhigang Wang a, b, c, d, 1, Fengzhi Wu a, c, d, * a
College of Horticulture, Northeast Agricultural University, Harbin 150030, PR China College of Life Science and Agroforestry, Qiqihar University, Qiqihar 161006, PR China Heilongjiang Provincial Key University Laboratory of Cold Area Vegetable Biology, Northeast Agricultural University, Mucai 59, Xiangfang, Harbin 150030, PR China d Ministry of Agriculture Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in Northeast China, Northeast Agricultural University, Mucai 59, Xiangfang, Harbin 150030, PR China b c
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a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 4 February 2015 Available online xxx
Pot experiments were performed to investigate the effects of companion cropping with D125 wheat on Fusarium wilt in watermelon. Fusarium oxysporum f. sp. niveum (Fon) is responsible for Fusarium wilt in watermelon. Also, the relationship between root exudates of wheat and watermelon growth was estimated. Studies showed that companion cropping with D125 wheat reduced the incidence rate of watermelon Fusarium wilt. Companion cropping with D125 wheat decreased malondialdehyde content and increased activities of phenylalanine ammonia-lyase and polyphenoloxidase and contents of flavonoid, total soluble phenolics and lignin in watermelon roots after inoculation with Fon compared to monoculture. qRT-PCR showed that the expression levels of six specific genes were higher during the early stage of Fon infection in companion cropping than in monoculture. D125 wheat root exudates increased root length, root surface area, root volume, root number, root dry weight, but decreased root mean diameter in watermelon seedlings in the absence of sodium orthovanadate. These results suggest that companion cropping with D125 wheat reduced Fusarium wilt in watermelon by promoting the growth of watermelon roots and by triggering gene expression and physiological changes to protect the watermelon from injury. © 2015 Published by Elsevier Ltd.
Keywords: Watermelon Companion crop Fusarium oxysporum f. sp. niveum D125 wheat root exudates Growth of watermelon roots Secondary metabolism compounds Gene expression
Introduction Every year thousands of hundred tons of watermelon were produced worldwide. However, the yield was affected because of
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Abbreviations: Fon, Fusarium oxysporum f. sp. niveum; MDA, Malondialdehyde; PPO, polyphenoloxidase; PAL, phenylalanine ammonia-lyase; PL, phospholipase; AOS, allene oxide synthase; DAHPS, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase; CCOMT, caffeovl-CoA O-methyltransferase; 4CL, 4-hydroxycinnamoyl-CoA ligase. * Corresponding author. Ministry of Agriculture Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in Northeast China, Northeast Agricultural University, Mucai 59, Xiangfang, Harbin 150030, PR China. Tel.: þ86 451 55190278. E-mail addresses: [email protected] (W. Xu), [email protected] (F. Wu). 1 Weihui Xu & Zhigang Wang contributed equally to this work, and are co-first authors.
the threat from Fusarium wilt [52]. Fusarium-mediated wilting of watermelon is caused by the pathogen Fusarium oxysporum f. sp. niveum (Fon) which is considered as the most important soilborne facultative parasite, which can lead to important loss economically, and limits production in many areas of the world [46]. The control of Fon depends mainly on fungicidal treatments [14], however, the use of fungicides may cause hazards to human health and may directly increase environmental pollution. Because of these associated problems, researchers have been trying to use some safer alternative methods for Fon control, one of which is intercropping [36]. Intercropping is a promising allelopathic approach in soil-borne disease management [17], it is adapted especially when some nonhost plant species are antagonistic to soil-borne pathogens. Some studies indicated that root exudation played great influences in growth of neighboring plants and microbes [11,44]. A little change in root exudates could result in huge alterations in the community
http://dx.doi.org/10.1016/j.pmpp.2015.02.003 0885-5765/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: Xu W, et al., Companion cropping with wheat increases resistance to Fusarium wilt in watermelon and the roles of root exudates in watermelon root growth, Physiological and Molecular Plant Pathology (2015), http://dx.doi.org/10.1016/ j.pmpp.2015.02.003
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structure of microorganisms in the rhizosphere [2,26]. Ren et al. [36] reported that intercropping with aerobic rice, a rice cultivated in unsaturated soil, could reduce Fusarium wilt disease in watermelon. Hao et al. [19] demonstrated that aerobic rice root exudates had a key role in controlling Fusarium wilt disease in watermelon. Wu et al. [45] reported that wheat allelopathy functioned in the management of weeds, pests, and diseases. Plant species or genotypes managed potential benefit to soil-borne diseases through root exudates [18]. Xu et al. [48] reported that companion cropping with D123 wheat could alleviate Fusarium wilt in watermelon through inhibiting the mycelia growth of Fon. In previous studies, we found that companion cropping with D125, a different wheat cultivar, could improve the watermelon growth [49,50]. However, D125 wheat root exudates could not inhibit mycelial growth of Fon [48]. So far, no reports have confirmed whether companion cropping with wheat can reduce the occurrence of Fusarium wilt in watermelon through improving watermelon growth. Also, the relationship between root exudates of wheat and watermelon growth should be evaluated to understand how the companion cropping can affect watermelon growth. Malondialdehyde (MDA), a decomposition end product of polyunsaturated fatty acids, and relative electrolytic conductivity, have been frequently utilized as suitable biomarkers for lipid peroxidation and membrane permeability in plants exposed to stressful environments [31]. The activities of polyphenoloxidase (PPO) and phenylalanine ammonia-lyases (PAL) are usually used to assess physiological and biochemical responses of plants to biotic and abiotic stresses [16]. Lignin, flavonoids and phenolics phytochemicals are the most important groups of secondary metabolites and had important role in inhibiting plant pathogens [24,40]. The jasmonic acid (JA) and shikimate-phenylpropanoid-lignin biosynthesis genes are relevant to watermelon responses to Fon1 infection [29]. By analyzing the changes of the above physiological parameters and gene expression under watermelonewheat companion cropping, we hope to understand the relationship between watermelon and Fon by using D125 wheat as companion crop. The objectives of this study were to study the following issues: (1) watermelon wilt disease in response to companion cropping with D125 wheat; (2) enzymatic activities related to plant resistance, the content of lignin, flavonoid and total soluble phenolics in response to Fon in watermelon roots under watermelon-wheat companion cropping; (3) the expressions of genes in the jasmonic acid and shikimate phenylpropanoid lignin biosynthesis pathways in response to Fon in watermelon roots under watermelon-wheat companion cropping; (4) the relationship between wheat root exudates and watermelon growth and the underlying mechanism of improving watermelon growth.
Plates were drenched with sterile distilled water and the conidia were carefully freed from the culture surface with a fine brush. Afterwards, the suspension was filtered through three layers of sterile cheese cloth to remove mycelial fragments. The conidia concentration was determined using the hemocytometer. Inoculation experiment All watermelon plants for inoculation experiments were grown in 70% relative humidity under a 14 h/10 h (light/dark, 28 C/18 C) photoperiod in a growth chamber. The experiments included two treatments: (i) watermelon monoculture (control) and (ii) companion cropping of watermelon with D125 wheat. All pots were arranged randomly with three replicates per treatment, in which each replicate had 60 plants to allow for destructive sampling. Watermelon seeds (Jingxin No.1) were surface-sterilized with 0.1% mercury for 10 min, rinsed four times in sterile distilled water, and sowed in a container which filled with steam-sterilized peat soil and vermiculite (v/v, 1:1). Seven days after seeding (when the first true leaf unfolded), the plantlets were gently transplanted into a plastic pot (13 cm in height and 15 cm in diameter) containing a moist sterilized mixture of peat soil and vermiculite (v/v, 1:1). In the companion cropping treatment, D125 wheat seeds were surfacesterilized with 5% (v/v) H2O2 for 30 min, washed with sterilized water, and then sowed directly on the side of the watermelon plant at the five-leaf stage, each pot contained 30 wheat seedlings and one watermelon seedling. Wheat seedlings were kept apart 5 cme7 cm from watermelon seedling. The wheat seedlings were cut for few times at 15 cm height during the experimental period to avoid shading of the watermelon and ensure good aeration. As the control, watermelon monoculture, each pot contained one watermelon seedling. The seedlings were watered every two days with Hoagland nutrient solution throughout the experiments [29]. Peat soil and vermiculite moisture levels were maintained by weighing irrigation manually. Twenty days later, the watermelon plants in both treatments were inoculated with conidia suspension of Fon (1.5 105conidia/g soil). The samples were collected at different time points, including pre-inoculation, 4 h, 8 h, 12 h, 1 d, 3 d, 5 d, 8 d, 10 d, and 15 d post-inoculation, respectively. The entire roots of 5 watermelon plants per replicate were harvested for each time point. Soil attached was washed off with running water, and the roots were rinsed thrice with distilled water. Afterwards, the watermelon roots were immediately frozen in liquid nitrogen, and kept at 80 C for analysis in the gene expression, the enzyme activities of PPO and PAL, as well as the content of MDA, lignin, flavonoid and total soluble phenolics. Assessment of disease incidence
Materials and methods Materials Watermelon seeds (Jingxin No.1), moderately susceptible to Fon, were purchased from the Golden Seed Company, Beijing, China. Wheat seeds (D125) was provided by Laboratory of Vegetable Physiological Ecology, Department of Horticulture, Northeast Agricultural University, Harbin, Heilongjiang Province. The F. oxysporum f. sp. niveum was recovered from a symptomatic watermelon plant in a greenhouse plot, Northeast Agricultural University, China, and identified as Fon1 and used in this study [1]. Conidia preparation Fon mycelium was grown in dish plates with PDA in the dark at 28 C for 10 days to induce sporulation as described previously [25].
Wilt incidence was assessed after inoculation for 15 days on 60 plants per replicate. The incidence was expressed as percentage, diseased plants over the total number of plants [47]. Determination of MDA content and enzymatic activities in watermelon roots The content of MDA in watermelon roots was determined using the thiobarbituric acid (TBA) coloration method [22]. MDA content was calculated from the difference in absorbance between 532 nm and 600 nm using the extinction coefficient of 155 mM1 cm1 and expressed as mmol g1 fresh weight. PAL activity was determined spectrophotometrically at 290 nm by determining the production of trans-cinnamic acid from Lphenylalanine [33]. PPO activity was measured by incubating the enzymatic extract for 1 min in sodium phosphate solution that
Please cite this article in press as: Xu W, et al., Companion cropping with wheat increases resistance to Fusarium wilt in watermelon and the roles of root exudates in watermelon root growth, Physiological and Molecular Plant Pathology (2015), http://dx.doi.org/10.1016/ j.pmpp.2015.02.003
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contained catechol, and then monitoring the change of absorbance spectrophotometrically at 398 nm [42]. Determination of total soluble phenolics in watermelon roots The experiment was conducted three times, with three replicates per treatment. Watermelon roots were collected, freeze-dried (in a Thermo LL3000) for 72 h, and then ground to a fine powder with a mortar and pestle, while liquid nitrogen was added. The fine powder was transferred to a plastic tube and stored at 80 C until further analysis. A representative sample of 0.1 g of the fine powder material from each replication and treatment was transferred to an Ependorf tube, homogenized with 1.5 ml of 80% methanol, and extracted overnight in a rotary shaker (150 rpm) at room temperature. The homogenate solution was protected from light oxidation by covering the Ependorf tube with aluminum foil. The dark-green methanolic extract was centrifuged at 12,000 g for 5 min, and the supernatant was transferred to a new Ependorf tube and stored at 20 C. The residue was kept at 20 C for further determination of lignin and lignin-like phenolic polymers. The methods developed by Zieslin and Ben-Zaken [53] were used to analyze the total soluble phenolics with a few modifications. A volume of 150 ml of 0.25 N Folin and Ciocalteau's Phenol reagent (SigmaeAldrich, USA) was added to 150 ml of methanolic extract and the mixture was homogenized and kept at room temperature for 5 min. Next, 150 ml of 1 M Na2CO3 was added to the mixture, which was homogenized again and allowed to stand at room temperature for 10 min. The mixture was further homogenized with 1 ml of distilled water and allowed to stand for 1 h at room temperature. The absorbance of a representative sample (500 ml) of the mixture from each replication and treatment was measured at 725 nm in a spectrophotometer. Total soluble phenolics were expressed as mg of phenolics (in terms of catechol) per g of dried root tissue. Determination of lignin in watermelon roots A 1.5 ml volume of sterile distilled water was added to the residue obtained after extraction of total soluble phenolics and after homogenization, the mixture was centrifuged at 12,000 g for 5 min. The supernatant was discarded and the residue was left to dry at 65 C overnight. The dried alcohol-insoluble residue, containing both true lignin and phenolic acids that were esterified to the cell walls, was used to determine lignin content [5]. A 1.5 ml volume of thioglycolic acid solution (1:10, v/v) and 2 N HCl was added to the dried residue. The Ependorf tube was shaken gently to hydrate the residue, and then placed in boiling water for 4 h. The tube was placed on ice in a cold room (4 C) for 10 min. The mixture was centrifuged at 12,000 g for 10 min, the supernatant was discarded, and the precipitate was washed with 1.5 ml of sterile distilled water and then centrifuged at 10,000 g for 10 min. After centrifugation, the supernatant was discarded, the precipitate was resuspended in 1.5 ml of 0.5 N NaOH, and the mixture was agitated overnight in a rotary shaker (150 rpm) at room temperature. The mixture was centrifuged at 10,000 g for 10 min, and the supernatant was transferred to a new Ependorf tube. After adding 200 ml of concentrated HCl to the supernatant, the Ependorf tube was transferred to a cold room (4 C) for 4 h to allow the ligninthioglycolic acid (LTGA) derivatives to precipitate. Following centrifugation at 10,000 g for 10 min, the supernatant was discarded, and the orange-brown precipitate was dissolved in 2 ml of 0.5 N NaOH. The absorbance of LTGA derivatives in the supernatant was measured at 280 nm in a spectrophotometer. The concentration of LTGA derivatives was expressed as mg g1 of dried root tissue by using lignin alkali, 2-hydroxypropyl ether (Sigma-Aldrich, USA) as a standard.
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Determination of flavonoid in watermelon roots The methods developed by Pirie and Mullins [35] were used to determine the flavonoid with a few modifications. A 0.5 g sample of watermelon roots was extracted from each replicate with 5 ml of 1% hydrochloric acid-methanol solutions for 24 h in the dark at room temperature. The 0.5 ml of extraction solution was diluted to 10 ml with 1% hydrochloric acid-methanol solution, and the diluted solution was determined spectrophometrically at 325 nm. The flavonoid contents were expressed as mg g1 fresh root tissue by using rutin (Sigma-Aldrich, USA) as a standard.
RNA isolation Total RNA was extracted from the roots and purified using a SK8661 Kit according to the manufacturer's instructions. RNA quality and quantity were determined using a spectrophotometer and agarose gel electrophoresis.
qRT-PCR An AMV First Strand cDNA Synthesis Kit synthesized the firststrand of cDNA using 5 ml of total RNA. This initial strand of cDNA was used as the template for qRT-PCR. The qRT-PCR reactions were run on an ABI StepOne plus real-time PCR system (StepOne, America). Each reaction (20 ml total volume) consisted of 1 ml of eightfold diluted cDNA, 1 ml of primer mix (10 mM of each forward and reverse primer), and 10 ml of SybrGreen qPCR Master Mix (2X). Individual reactions were run with each primer pair at annealing temperatures of 60 C. PCR cycling conditions were as follows: 95 C for 2 min, 40 cycles of 95 C for 10 s, and 60 C for 40 s. Genespecific primers for qRT-PCR were designed based on the EST sequences for six genes, namely: PL (phospholipase), AOS (allene oxide synthase), DAHPS (3-deoxy-D-arabino-heptulosonate-7phosphate synthase), PAL (phenylalanine ammonia-lyase), 4CL (4hydroxycinnamoyl-CoA) and CCOMT (caffeovl-CoA-O-methyltransferase). Table 1 summarizes the specific sets of primers used for the amplification of each cDNA. Melting curves were performed at the end of each reaction run to detect primer dimers and secondary products. The conditions were 95 C for 15 s, followed by 60 C for 1 min, and then kept at 95 C. The watermelon 18S rRNA gene was used as an internal control. The calculation of relative gene expression was conducted as described by Livak and Schmittgen [27]. Each measurement was replicated three times.
Table 1 Genes and primers used in real-time qRT-PCR analysis. Gene
Forward primer
Reverse primer
18S rRNA PL
50 -ATCAGAAAGTAGCA CAACAAGCAC-30 50 -ATTCAGAAATGACGAT GTGGC-30 50 -CGAGAAGGCGAGAGA TGAAGA-30 50 -ATTCGTGATACCTTC CGTGTTC-30 50 -CGAGTTTGGGTTGC CATTTAT-30 50 -CTACATTTTTGGTTGC TCTCTGC-30 50 -GTTTTGGGTGGGTGA TAAGAAG-30
50 -CTTCCGTTCAGCCTTTACCAT-30
AOS DAHPS CCOMT PAL 4CL
50 -TAAGGCGAGCAGATAAGGGAT-30 50 -GCTGAAACACGTGCTATGGTC-30 50 -CTGCCATTCTTCCTACCTTGAT-30 50 -CTCCACCTATCTTCACGAGTTTTTA-30 50 -AAATCTTGAGGGGTGAAGTGTG-30 50 -GGACTATTGATAAAGAAGGATGGC-30
Note: The watermelon 18S rRNA gene was used as an internal control.
Please cite this article in press as: Xu W, et al., Companion cropping with wheat increases resistance to Fusarium wilt in watermelon and the roles of root exudates in watermelon root growth, Physiological and Molecular Plant Pathology (2015), http://dx.doi.org/10.1016/ j.pmpp.2015.02.003
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Assessment of the effects of wheat root exudates on watermelon root growth To assess the effects of wheat root exudates on watermelon root growth, sodium orthovanadate, an inhibitor of root exudates [28], was applied to the gel system [13], consisting of a watermelon seedling planted in agar that had previously contained the D125 wheat as donor plant or without the wheat as control. There were four treatments: 1) D125 (planted D125 wheat in gel system), 2) CK (only planted watermelon), 3) ZD125 (planted D125 wheat and added 30 mmol/L sodium orthovanadate, an inhibitor for root exudate, in gel system) and 4) ZCK (only planted watermelon and added 30 mmol/L sodium orthovanadate in gel system). Triplicates were done for each treatment. D125 wheat seeds were surface sterilized with 0.1% mercuric chloride for 8 min, and rinsed five times with sterile water. The sterilized D125 wheat seeds were planted in petri dish, which contained half-strength Hoagland media, in the dark at 22 C for 48 h to germinate. In D125 and ZD125 treatments, the germinated 25 D125 wheat seeds were transplanted to 250 ml beaker, filled with transparent growth medium consisting of half-strength Hoagland solution and 1.5% phytagel (pH ¼ 5.8). Aluminum foil was used to cover the gel surface and wrap the beaker to keep the roots away from light. Wheat plants were grown at a relative humidity of 70% under a 16/8 h (light/ dark, 22 C/18 C) photoperiod in a growth chamber. The wheat seedlings were pulled out after 7 days growing (the root exudates was still kept in gel system), and then one germinated and sterile watermelon seed was transplanted to beaker. At the same time, one germinated and sterile watermelon seed was transplanted to beaker in CK and ZCK treatments. Watermelon plants were grown at a relative humidity of 70% under a 14/10 h (light/dark, 28 C/ 18 C) photoperiod in a growth chamber. Watermelon roots were harvested on the 30th days after germination, and the harvested watermelon roots were scanned and analyzed root length, root surface area, root volume, root mean diameter, number of roots by root analyzer (LA-S2400) [9].
Fig. 1. Effects of companion cropping with wheat on Fusarium wilt infection in watermelon seedlings. Note: incidence rate of Fusarium wilt in watermelon 15 days after Fon inoculation in sterilized turf soil and vermiculite (1:1, v/v) in growth chamber. Significant differences between treatments were indicated by different letters (P 0.05, independent samples T test). CK: monoculture of watermelon; D125: D125 wheat as companion crop.
accompanied with D125 wheat than in monoculture (Fig. 2). Compared with the monoculture system, companion cropping with wheat increased the activities of PPO by 24.8% in watermelon roots 15 days after Fon inoculation. However, 5 and 10 days after Fon inoculation, no significant difference of PPO activity in the roots of the two cropping systems could be detected (Fig. 2). PAL activity was no difference in watermelon roots for both cropping systems before Fon inoculation. However, PAL activity was higher significantly in watermelon roots of companion cropping than in monoculture after Fon inoculation for 5 days or longer (Fig. 2). Changes of total soluble phenolics, lignin and flavonoid in watermelon roots after exposured to Fon under watermelon-wheat companion cropping
Statistical analyses All data were expressed as mean ± standard error and subjected to ANOVA. The data were analyzed by t-tests method (P 0.05) using the SAS statistical software. Results Responses of companion cropping system to watermelon Fusarium wilt Watermelon plants began to develop symptoms of Fusarium wilt 15 days after inoculation with Fon. However, the incidence rate of watermelon Fusarium wilt was 46.4% in the monoculture system, while the incidence rate of Fusarium wilt in watermelons accompanied with D125 wheat was 13.3% (P < 0.05, independent samples T test), it was significantly lower than monoculture (Fig. 1). Changes of MDA content and enzymatic activities in watermelon roots exposured to Fon under watermelon-wheat companion cropping Before Fon inoculation, MDA (Malondialdehyde) content of the watermelon roots was similar for both the monoculture and the companion cropping with D125 wheat. After Fon inoculation for 5 days, MDA contents started to increase gradually in the roots of watermelon for both cropping systems. However, the increase of MDA content was significantly lower in watermelon roots
Without Fon inoculation, there was no significant difference in total soluble phenolics in watermelon roots for both cropping systems. Total soluble phenolics was higher clearly in watermelon roots of companion cropping than in monoculture at 10 days and 15 days after Fon inoculation (Fig. 2). Before Fon inoculation, the lignin contents were similar in the roots of watermelon for both in monoculture and in companion cropping with D125 wheat. The lignin contents in companion cropping with D125 wheat were significantly higher (22.0%) than in monoculture at 10 days after Fon inoculation. Compared with the monoculture system, lignin content increased up to 21.9% in companion cropping with D125 wheat at 15 days after Fon inoculation (Fig. 2). No significant difference of flavonoid contents could be detected between preinoculation and 10 days post-inoculation. However, flavonoid contents were 54.2% higher in D125 companion system than in monoculture at 15 days after Fon inoculation (Fig. 2). Responses of gene expressions involved in JA biosynthesis and shikimate-phenylpropanoid-ligin pathways to Fon This study analyzed the expression of genes in JA biosynthesis and shikimate-phenylpropanoid-lignin pathways, which were related in the watermelon roots under companion cropping and monoculture systems after challenging by Fon [29]. Phospholipase (PL) and allene oxide synthase (AOS) are involved in the different steps of the JA biosynthesis pathway. Results from qRT-PCR showed that the expressions of PL and AOS were significantly increased at 4,
Please cite this article in press as: Xu W, et al., Companion cropping with wheat increases resistance to Fusarium wilt in watermelon and the roles of root exudates in watermelon root growth, Physiological and Molecular Plant Pathology (2015), http://dx.doi.org/10.1016/ j.pmpp.2015.02.003
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Fig. 2. Responses of MDA content, PAL activity, PPO activity, flavonoid content, total soluble phenolics and lignin content in watermelon roots to the presence of wheat as companion crop and to infection with Fon. MDA: Malondialdehyde; PAL: Phenylalanine ammonia-lyase; PPO: Polyphenoloxidase; CK: monoculture of watermelon; D125: D125 wheat as companion crop; Pre-: before Fon inoculation; Post-5 d: 5 days after Fon inoculation; Post-10 d: 10 days after Fon inoculation; Post-15 d: 15 days after Fon inoculation.
8 and 12 h after Fon inoculation in the companion cropping system. However, in the monoculture system, expressions of PL and AOS genes were increased slightly at 24 h after Fon inoculation (Fig. 3). Expressions of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS), phenylalanine ammonia-lyase (PAL), 4-hydroxycinnamoyo-CoA ligase (4CL), and caffeovl-CoA O-methyltransferase (CCOMT), which involved in different steps of the shikimatephenylpropanoid-lignin biosynthesis pathway, were also analyzed. DAHPS expression was upregulated at 1 and 5 days after inoculation in the companion cropping system, whereas its expression was upregulated at 3 and 8 days after inoculation in
monoculture. PAL expression level was higher at 1 and 8 days after inoculation in companion cropping than in monoculture system. 4CL expression was upregulated at 1 and 8 days after inoculation in the companion cropping system, whereas its expression level was lower at 3 and 5 days after inoculation in the companion cropping system than in monoculture system. CCOMT expression was induced at 1, 3, 5 and 8 days after inoculation in companion cropping whereas CCOMT expression was similar to the preinoculation levels at these time point in the monoculture (Fig. 4). Effect of D125 wheat root exudates on root growth of watermelon seedlings The root length, root surface area, root volume, root dry weight and root numbers of watermelon seedlings in the D125 treatment in the gel system (D125) were higher than those in the control (CK), whereas root mean diameter for watermelon seedlings was thinner compared to CK (Fig. 5). Interestingly, the effect was abolished while sodium orthovanadate added in gel system, there were no significant differences on root length, root surface area, root volume, root number, root mean diameter and root dry weight in watermelon seedlings in ZD125 and ZCK (Fig. 6). Discussion
Fig. 3. Expressions of PL and AOS genes involved in JA biosynthesis in watermelon when grown alone (CK) or together with wheat variety D125 (D125) before and after inoculation with Fon. Results from qRT-PCR. The JA biosynthetic pathway was adopted from Creelman and Mullet [10]. PL: phospholipase; AOS: allene oxide synthase; Pre-: before Fon inoculation; Post-4 h: 4 h after Fon inoculation; Post-8 h: 8 h after Fon inoculation; Post-12 h: 12 h after Fon inoculation; Post-24 h: 24 h after Fon inoculation.
Some findings showed the importance of root exudates in defending the plant against pathogenic microorganisms [38,51]. Hao et al. [19] demonstrated the mechanism in which rice root exudates inhibited spore germination and sporulation of Fon in a watermelon/aerobic rice intercropping system. In previous study, we found that there was no significant difference in mycelium growth of Fon with or without the D125 wheat root exudates [48]. However, this experiment showed that watermelon seedlings could resist against Fusarium wilt in the companion cropping with D125 wheat system as compared to monoculture (Fig. 1). The reasons
Please cite this article in press as: Xu W, et al., Companion cropping with wheat increases resistance to Fusarium wilt in watermelon and the roles of root exudates in watermelon root growth, Physiological and Molecular Plant Pathology (2015), http://dx.doi.org/10.1016/ j.pmpp.2015.02.003
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Fig. 4. Expressions of some genes involved in shikimate-phenylpropanoid-lignin biosynthesis in companion cropping and monoculture interactions between watermelon and Fon via qRT-PCR. The shikimate-phenylpropanoid-lignin biosynthetic pathway was adopted from Whetten and Sederoff [43]. DAHPS: 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase; PAL: phenylalanine ammonia-lyase; 4CL: 4-hydroxycinnamoyl-CoA ligase; CCOMT: caffeovl-CoA-O-methyltransferase. Pre-: before Fon inoculation; Post-1 d: 1 d after Fon inoculation; Post-3 d: 3 d after Fon inoculation; Post-5 d: 5 d after Fon inoculation; Post-8 d: 8 d after Fon inoculation.
may be that D125 wheat root exudates promoted the growth of watermelon roots (Fig. 5). When plants are attacked by pathogens or placed under some other stress, a series of physiological and biochemical responses could occur in the plants [8,15]. MDA content represents the degree of damage to plant cell membranes by the stress period, the higher the level of MDA is, the more severe the damage to the cell [32]. This experiment showed that companion cropping with D125 wheat decreased MDA content in watermelon roots after inoculation with Fon compared with monoculture (Fig. 2). This change of content in MDA implied that companion cropping with D125 wheat increased
the stability of biological membranes to prevent infection of watermelon from Fon. In this study, companion cropping with D125 wheat significantly increased the PPO activities in watermelon roots compared with monoculture (Fig. 2). Due to the increase of PPO activity the more lignins and other phenolics were synthesized to make cell walls stronger, so that it prevented invasion from pathogen [23,37]. Hassan et al. [20] reported that the resistance against bacterial wilt of potato was induced by application of plant extracts, which was associated with the increase of PAL activities. In our investigation, PAL activity in watermelon roots was higher at 5, 10, 15 days after
Fig. 5. The effect of wheat root exudates on root length, root surface area, root volume, root mean diameter, number of roots, root dry weight in watermelon seedlings. CK: only planted watermelon in gel system where was not added sodium orthovanadate; D125: D125 wheat were planted in gel system where was not added sodium orthovanadate, wheat seedlings were pulled out after 7 days growing, and then a watermelon seedling was planted in gel system.
Please cite this article in press as: Xu W, et al., Companion cropping with wheat increases resistance to Fusarium wilt in watermelon and the roles of root exudates in watermelon root growth, Physiological and Molecular Plant Pathology (2015), http://dx.doi.org/10.1016/ j.pmpp.2015.02.003
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Fig. 6. The effect of wheat root exudates on root length, root surface area, root volume, number of roots, root mean diameter, root dry weight in watermelon seedlings after adding root exudates in the gel system. ZCK: only planted watermelon in gel system where was added sodium orthovanadate; ZD125: D125 wheat were planted in gel system where was added sodium orthovanadate, wheat seedlings were pulled out after 7 days growing, and then a watermelon seedling was planted in gel system.
Fon inoculation in companion cropping with D125 wheat compared with monoculture. This implied that D125 wheat root exudates could induce resistance against Fusarium wilt in watermelon and delay the occurrence of Fusarium wilt in watermelon. Nicholson and Hammerschmidt [34] reported that phenolic compounds formed in response to infection with the pathogen were considered as part of an active defense response and consequently protected plants against pathogenic attacks. Phenolic compounds could play a major role in wilt disease resistance through the inactivation of fungal cell wall degrading enzymes (cellulase and pectin methyl esterase), thereby restricting the degradation of the cell wall and the fungal invasion in plants [30]. In the present work, there was a noticeable increase in total phenolic compounds in watermelon roots at 10 and 15 days after Fon inoculation in companion cropping with D125 wheat compared with monoculture (Fig. 2). It is suggested that companion with D125 wheat enhanced resistance in watermelon to Fusarium wilt by increasing total phenolic compounds in watermelon roots. Flavonoids are a broad group of secondary metabolites and have been reported to confer disease resistance in different plant species [3]. Lignin, a major component of cell walls of vascular plants, was considered as a first line defense against penetration of invasive pathogens [6]. At 15 days after Fon inoculation, flavonoid content in watermelon roots were higher in companion cropping with D125 wheat than in monoculture (Fig. 2). At 10 and 15 days after Fon inoculation, lignin content was higher in watermelon roots in companion cropping with D125 wheat than in monoculture. It implied that companion with D125 wheat prevented invasion of pathogens by increasing the content of secondary metabolism. Interestingly, the content of MDA became lower in companion cropping system at 5 days after Fon inoculation, compared to the control. It suggested that the damage of membrane was less in companion cropping system than in monoculture. Thus, increasing of the membranes stability was a key factor for reducing Fusarium wilt in watermelon in companion cropping system. The compounds generated from phenylpropanoid and jasmonate pathways play the major roles in plant defense responses against pathogen attack [12,39]. Lü et al. [29] demonstrated that JA
biosynthesis and shikimate-phenylpropanoid-lignin pathways are important processes in watermelon resistance against Fon infection. We found in this study, expression levels of the genes in responsible for biosynthesis of JA and shikimate-phenylpropanoidlignin were enhanced in both monoculture and companion cropping with Fon inoculation. However, the timing and levels of the expressions were different between the two cropping system. In the companion cropping system, PL and AOS gene expression were strongly enhanced at 4 h, 8 h and 12 h after Fon inoculation and the levels were higher than in the monoculture system (Fig. 3). The expression level of DAHPS gene was higher in companion cropping than in monoculture at 1 d and 5 d after Fon inoculation. The expression levels of PAL and 4CL gene were higher in companion cropping than in monoculture at 1 d and 8 d after Fon infection, also the CCOMT gene expression level was strongly upregulated in companion cropping at 1, 3, 5 and 8 days after Fon inoculation (Fig. 4). Our results showed that the timing points for the gene expression were earlier than the other indicators, and the response was stronger in companion cropping with D125 wheat than in monoculture. Therefore, we speculate that the resistance against Fon was increased in companion cropping system by the high expression of the six genes after inoculated with Fon. In the previous study, we found that companion cropping wheat promoted growth of watermelon [49,50]. The phytochemicals presented in the root exudates of plants mediated several types of communication processes in the rhizosphere such as rooteroot, root-microbe and rooteinsect interactions [4,41]. Loyala-Vargas et al. [28] demonstrated that the exudation of some compounds in Arabidopsis root was inhibited by sodium orthovanadate. In this study, we compared treatments with and without the addition of sodium orthovanadate to the gel system to evaluate the role of wheat root exudates in promoting watermelon root growth. It was observed that D125 wheat root exudates have significant positive effects on the growth of watermelon roots (Fig. 5). When sodium orthovanadate was added in the gel system, D125 wheat root exudates (ZD125) had no effect on the growth of watermelon roots, which suggests that the exudation of some compounds in D125 wheat root were inhibited (Fig. 6). This study indicated that D125
Please cite this article in press as: Xu W, et al., Companion cropping with wheat increases resistance to Fusarium wilt in watermelon and the roles of root exudates in watermelon root growth, Physiological and Molecular Plant Pathology (2015), http://dx.doi.org/10.1016/ j.pmpp.2015.02.003
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wheat root exudates had an important role in promoting the growth of watermelon roots. These results support the role of root exudates in the phenomenon that companion cropping with D125 wheat could increase growth of watermelon. Caffaro et al. [7] evaluated the role of secondary metabolites in the development of root architecture by comparison of treatments between without and with the addition of activated charcoal to cropping systems, and indicated that secondary metabolites presented in the root exudates played key roles on the development of root architecture. Logically, natural plant growth regulators may be involved in root facilitation interactions [21], even if we did not identify what they are still. Further studies are needed to define the functional components in D125 wheat root exudates that promote the growth of watermelon root. In the conclusion, companion cropping with D125 wheat could reduce disease incidence of Fusarium wilt in watermelon by promoting growth of watermelon roots and inducing gene expression and physiological changes to protect watermelon from the infection. Further research is needed to identify the functional components in D125 wheat root exudates that promote watermelon roots growth, and also it is needed to investigate the effects of companion cropping with D125 wheat on the microbial community diversity in the rhizosphere soil with watermelons in the field experiments. Acknowledgments We gratefully acknowledge National Nature Science Foundation of China (31471917) and Science and Technology Research Project of Heilongjiang Province Department of Education, China (12541859) for theirs funding of this project. References [1] An MJ, Wu FZ, Liu B. Study on the differentiation of physiological race from Fusarium oxysporum f.sp.niveum and the resistance of different watermelon cultivars in Heilongjiang. J Shanghai Jiaot Univ 2009;27(5):494e500 [in China]. [2] Badri DV, Vivanco JM. Regulation and function of root exudates. Plant Cell Environ 2009;32:666e81. [3] Bahraminejad S, Asenstorfer RE, Riley IT, Schutz CJ. Analysis of antimicrobial activity of flavonoids and saponins isolated from the shoots of oats (Avena sativa L.). J Phytopathol 2008;156:1e7. [4] Bais HP, Park SW, Weir T, Callaway RM, Vivanco JM. How plants communicate using the underground information superhighway. Trends Plant Sci 2004;9: 26e32. [5] Barber MS, Ride JP. A quantitative assay for induced lignifications in wounded wheat leaves and its use to survey potential elicitors of the response. Physiol Mol Plant P 1988;32(2):185e97. [6] Bhuiyan NH, Selvaraj G, Wei YD, King J. Role of lignifications in plant defense. Plant Signal Behav 2009;4(2):158e9. [7] Caffaro MM, Vivanco JM, Gutierrez Boem FH, Rubio G. The effect of root exudates on root architecture in Arabidopsis thaliana. Plant Growth Regul 2011;64:241e9. langer RR, Benhamou N, Paulitz TC. Defense enzymes induced in [8] Chen C, Be cucumber roots by treatment with plant growth-promoting rhizobacteria (PGPR) and Pythium aphanidermatum. Physiol Mol Plant P 2000;56:13e23. [9] Chu LR, Pan K, Wu FZ, Tao L, Wang Y. Effects of Hexadecanoic acid on Fusarium oxysporum f. sp. niveum control and on growth of watermelon. Allelopathy J 2014;34(2):241e52. [10] Creelman RA, Mullet JE. Jasmonic acid distribution and action in plants: Regulation during development and response to biotic and abiotic stress. Proc Natl Acad Sci U S A 1995;92:4114e9. [11] De-la-Pena C, Lei Z, Watson BS, Sumner LW, Vivanco JM. Root-microbe communication through protein secretion. J Biol Chem 2008;283:25247e55. [12] Dixon RA, Paiva NL. Stress-induced phenylpropanoid metabolism. Plant Cell 1995;7:1085e97. [13] Fang SQ, Gao X, Deng Y, Chen XP, Liao H. Crop root behavior coordinates phosphorus status and neighbors: from field studies to three-dimensional in situ reconstruction of root system architecture. Plant Physiol 2011;155: 1277e85. [14] Fravel DR, Deahl KL, Stommel JR. Compatibility of the biocontrol fungus Fusarium oxysporum strain CS-20 with selected fungicides. Biol Control 2005;34:165e9. s JA, Jime nez-Díaz RM, Tena M. In[15] Garcia-Limones C, Herv as A, Navas-Corte duction of an antioxidant enzyme system and other oxidative stress markers
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Contents lists available at ScienceDirect
Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp
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Companion cropping with wheat increases resistance to Fusarium wilt in watermelon and the roles of root exudates in watermelon root growth
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Weihui Xu a, b, c, d, 1, Zhigang Wang a, b, c, d, 1, Fengzhi Wu a, c, d, * a
College of Horticulture, Northeast Agricultural University, Harbin 150030, PR China College of Life Science and Agroforestry, Qiqihar University, Qiqihar 161006, PR China Heilongjiang Provincial Key University Laboratory of Cold Area Vegetable Biology, Northeast Agricultural University, Mucai 59, Xiangfang, Harbin 150030, PR China d Ministry of Agriculture Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in Northeast China, Northeast Agricultural University, Mucai 59, Xiangfang, Harbin 150030, PR China b c
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a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 4 February 2015 Available online xxx
Pot experiments were performed to investigate the effects of companion cropping with D125 wheat on Fusarium wilt in watermelon. Fusarium oxysporum f. sp. niveum (Fon) is responsible for Fusarium wilt in watermelon. Also, the relationship between root exudates of wheat and watermelon growth was estimated. Studies showed that companion cropping with D125 wheat reduced the incidence rate of watermelon Fusarium wilt. Companion cropping with D125 wheat decreased malondialdehyde content and increased activities of phenylalanine ammonia-lyase and polyphenoloxidase and contents of flavonoid, total soluble phenolics and lignin in watermelon roots after inoculation with Fon compared to monoculture. qRT-PCR showed that the expression levels of six specific genes were higher during the early stage of Fon infection in companion cropping than in monoculture. D125 wheat root exudates increased root length, root surface area, root volume, root number, root dry weight, but decreased root mean diameter in watermelon seedlings in the absence of sodium orthovanadate. These results suggest that companion cropping with D125 wheat reduced Fusarium wilt in watermelon by promoting the growth of watermelon roots and by triggering gene expression and physiological changes to protect the watermelon from injury. © 2015 Published by Elsevier Ltd.
Keywords: Watermelon Companion crop Fusarium oxysporum f. sp. niveum D125 wheat root exudates Growth of watermelon roots Secondary metabolism compounds Gene expression
Introduction Every year thousands of hundred tons of watermelon were produced worldwide. However, the yield was affected because of
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Abbreviations: Fon, Fusarium oxysporum f. sp. niveum; MDA, Malondialdehyde; PPO, polyphenoloxidase; PAL, phenylalanine ammonia-lyase; PL, phospholipase; AOS, allene oxide synthase; DAHPS, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase; CCOMT, caffeovl-CoA O-methyltransferase; 4CL, 4-hydroxycinnamoyl-CoA ligase. * Corresponding author. Ministry of Agriculture Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in Northeast China, Northeast Agricultural University, Mucai 59, Xiangfang, Harbin 150030, PR China. Tel.: þ86 451 55190278. E-mail addresses: [email protected] (W. Xu), [email protected] (F. Wu). 1 Weihui Xu & Zhigang Wang contributed equally to this work, and are co-first authors.
the threat from Fusarium wilt [52]. Fusarium-mediated wilting of watermelon is caused by the pathogen Fusarium oxysporum f. sp. niveum (Fon) which is considered as the most important soilborne facultative parasite, which can lead to important loss economically, and limits production in many areas of the world [46]. The control of Fon depends mainly on fungicidal treatments [14], however, the use of fungicides may cause hazards to human health and may directly increase environmental pollution. Because of these associated problems, researchers have been trying to use some safer alternative methods for Fon control, one of which is intercropping [36]. Intercropping is a promising allelopathic approach in soil-borne disease management [17], it is adapted especially when some nonhost plant species are antagonistic to soil-borne pathogens. Some studies indicated that root exudation played great influences in growth of neighboring plants and microbes [11,44]. A little change in root exudates could result in huge alterations in the community
http://dx.doi.org/10.1016/j.pmpp.2015.02.003 0885-5765/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: Xu W, et al., Companion cropping with wheat increases resistance to Fusarium wilt in watermelon and the roles of root exudates in watermelon root growth, Physiological and Molecular Plant Pathology (2015), http://dx.doi.org/10.1016/ j.pmpp.2015.02.003
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structure of microorganisms in the rhizosphere [2,26]. Ren et al. [36] reported that intercropping with aerobic rice, a rice cultivated in unsaturated soil, could reduce Fusarium wilt disease in watermelon. Hao et al. [19] demonstrated that aerobic rice root exudates had a key role in controlling Fusarium wilt disease in watermelon. Wu et al. [45] reported that wheat allelopathy functioned in the management of weeds, pests, and diseases. Plant species or genotypes managed potential benefit to soil-borne diseases through root exudates [18]. Xu et al. [48] reported that companion cropping with D123 wheat could alleviate Fusarium wilt in watermelon through inhibiting the mycelia growth of Fon. In previous studies, we found that companion cropping with D125, a different wheat cultivar, could improve the watermelon growth [49,50]. However, D125 wheat root exudates could not inhibit mycelial growth of Fon [48]. So far, no reports have confirmed whether companion cropping with wheat can reduce the occurrence of Fusarium wilt in watermelon through improving watermelon growth. Also, the relationship between root exudates of wheat and watermelon growth should be evaluated to understand how the companion cropping can affect watermelon growth. Malondialdehyde (MDA), a decomposition end product of polyunsaturated fatty acids, and relative electrolytic conductivity, have been frequently utilized as suitable biomarkers for lipid peroxidation and membrane permeability in plants exposed to stressful environments [31]. The activities of polyphenoloxidase (PPO) and phenylalanine ammonia-lyases (PAL) are usually used to assess physiological and biochemical responses of plants to biotic and abiotic stresses [16]. Lignin, flavonoids and phenolics phytochemicals are the most important groups of secondary metabolites and had important role in inhibiting plant pathogens [24,40]. The jasmonic acid (JA) and shikimate-phenylpropanoid-lignin biosynthesis genes are relevant to watermelon responses to Fon1 infection [29]. By analyzing the changes of the above physiological parameters and gene expression under watermelonewheat companion cropping, we hope to understand the relationship between watermelon and Fon by using D125 wheat as companion crop. The objectives of this study were to study the following issues: (1) watermelon wilt disease in response to companion cropping with D125 wheat; (2) enzymatic activities related to plant resistance, the content of lignin, flavonoid and total soluble phenolics in response to Fon in watermelon roots under watermelon-wheat companion cropping; (3) the expressions of genes in the jasmonic acid and shikimate phenylpropanoid lignin biosynthesis pathways in response to Fon in watermelon roots under watermelon-wheat companion cropping; (4) the relationship between wheat root exudates and watermelon growth and the underlying mechanism of improving watermelon growth.
Plates were drenched with sterile distilled water and the conidia were carefully freed from the culture surface with a fine brush. Afterwards, the suspension was filtered through three layers of sterile cheese cloth to remove mycelial fragments. The conidia concentration was determined using the hemocytometer. Inoculation experiment All watermelon plants for inoculation experiments were grown in 70% relative humidity under a 14 h/10 h (light/dark, 28 C/18 C) photoperiod in a growth chamber. The experiments included two treatments: (i) watermelon monoculture (control) and (ii) companion cropping of watermelon with D125 wheat. All pots were arranged randomly with three replicates per treatment, in which each replicate had 60 plants to allow for destructive sampling. Watermelon seeds (Jingxin No.1) were surface-sterilized with 0.1% mercury for 10 min, rinsed four times in sterile distilled water, and sowed in a container which filled with steam-sterilized peat soil and vermiculite (v/v, 1:1). Seven days after seeding (when the first true leaf unfolded), the plantlets were gently transplanted into a plastic pot (13 cm in height and 15 cm in diameter) containing a moist sterilized mixture of peat soil and vermiculite (v/v, 1:1). In the companion cropping treatment, D125 wheat seeds were surfacesterilized with 5% (v/v) H2O2 for 30 min, washed with sterilized water, and then sowed directly on the side of the watermelon plant at the five-leaf stage, each pot contained 30 wheat seedlings and one watermelon seedling. Wheat seedlings were kept apart 5 cme7 cm from watermelon seedling. The wheat seedlings were cut for few times at 15 cm height during the experimental period to avoid shading of the watermelon and ensure good aeration. As the control, watermelon monoculture, each pot contained one watermelon seedling. The seedlings were watered every two days with Hoagland nutrient solution throughout the experiments [29]. Peat soil and vermiculite moisture levels were maintained by weighing irrigation manually. Twenty days later, the watermelon plants in both treatments were inoculated with conidia suspension of Fon (1.5 105conidia/g soil). The samples were collected at different time points, including pre-inoculation, 4 h, 8 h, 12 h, 1 d, 3 d, 5 d, 8 d, 10 d, and 15 d post-inoculation, respectively. The entire roots of 5 watermelon plants per replicate were harvested for each time point. Soil attached was washed off with running water, and the roots were rinsed thrice with distilled water. Afterwards, the watermelon roots were immediately frozen in liquid nitrogen, and kept at 80 C for analysis in the gene expression, the enzyme activities of PPO and PAL, as well as the content of MDA, lignin, flavonoid and total soluble phenolics. Assessment of disease incidence
Materials and methods Materials Watermelon seeds (Jingxin No.1), moderately susceptible to Fon, were purchased from the Golden Seed Company, Beijing, China. Wheat seeds (D125) was provided by Laboratory of Vegetable Physiological Ecology, Department of Horticulture, Northeast Agricultural University, Harbin, Heilongjiang Province. The F. oxysporum f. sp. niveum was recovered from a symptomatic watermelon plant in a greenhouse plot, Northeast Agricultural University, China, and identified as Fon1 and used in this study [1]. Conidia preparation Fon mycelium was grown in dish plates with PDA in the dark at 28 C for 10 days to induce sporulation as described previously [25].
Wilt incidence was assessed after inoculation for 15 days on 60 plants per replicate. The incidence was expressed as percentage, diseased plants over the total number of plants [47]. Determination of MDA content and enzymatic activities in watermelon roots The content of MDA in watermelon roots was determined using the thiobarbituric acid (TBA) coloration method [22]. MDA content was calculated from the difference in absorbance between 532 nm and 600 nm using the extinction coefficient of 155 mM1 cm1 and expressed as mmol g1 fresh weight. PAL activity was determined spectrophotometrically at 290 nm by determining the production of trans-cinnamic acid from Lphenylalanine [33]. PPO activity was measured by incubating the enzymatic extract for 1 min in sodium phosphate solution that
Please cite this article in press as: Xu W, et al., Companion cropping with wheat increases resistance to Fusarium wilt in watermelon and the roles of root exudates in watermelon root growth, Physiological and Molecular Plant Pathology (2015), http://dx.doi.org/10.1016/ j.pmpp.2015.02.003
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contained catechol, and then monitoring the change of absorbance spectrophotometrically at 398 nm [42]. Determination of total soluble phenolics in watermelon roots The experiment was conducted three times, with three replicates per treatment. Watermelon roots were collected, freeze-dried (in a Thermo LL3000) for 72 h, and then ground to a fine powder with a mortar and pestle, while liquid nitrogen was added. The fine powder was transferred to a plastic tube and stored at 80 C until further analysis. A representative sample of 0.1 g of the fine powder material from each replication and treatment was transferred to an Ependorf tube, homogenized with 1.5 ml of 80% methanol, and extracted overnight in a rotary shaker (150 rpm) at room temperature. The homogenate solution was protected from light oxidation by covering the Ependorf tube with aluminum foil. The dark-green methanolic extract was centrifuged at 12,000 g for 5 min, and the supernatant was transferred to a new Ependorf tube and stored at 20 C. The residue was kept at 20 C for further determination of lignin and lignin-like phenolic polymers. The methods developed by Zieslin and Ben-Zaken [53] were used to analyze the total soluble phenolics with a few modifications. A volume of 150 ml of 0.25 N Folin and Ciocalteau's Phenol reagent (SigmaeAldrich, USA) was added to 150 ml of methanolic extract and the mixture was homogenized and kept at room temperature for 5 min. Next, 150 ml of 1 M Na2CO3 was added to the mixture, which was homogenized again and allowed to stand at room temperature for 10 min. The mixture was further homogenized with 1 ml of distilled water and allowed to stand for 1 h at room temperature. The absorbance of a representative sample (500 ml) of the mixture from each replication and treatment was measured at 725 nm in a spectrophotometer. Total soluble phenolics were expressed as mg of phenolics (in terms of catechol) per g of dried root tissue. Determination of lignin in watermelon roots A 1.5 ml volume of sterile distilled water was added to the residue obtained after extraction of total soluble phenolics and after homogenization, the mixture was centrifuged at 12,000 g for 5 min. The supernatant was discarded and the residue was left to dry at 65 C overnight. The dried alcohol-insoluble residue, containing both true lignin and phenolic acids that were esterified to the cell walls, was used to determine lignin content [5]. A 1.5 ml volume of thioglycolic acid solution (1:10, v/v) and 2 N HCl was added to the dried residue. The Ependorf tube was shaken gently to hydrate the residue, and then placed in boiling water for 4 h. The tube was placed on ice in a cold room (4 C) for 10 min. The mixture was centrifuged at 12,000 g for 10 min, the supernatant was discarded, and the precipitate was washed with 1.5 ml of sterile distilled water and then centrifuged at 10,000 g for 10 min. After centrifugation, the supernatant was discarded, the precipitate was resuspended in 1.5 ml of 0.5 N NaOH, and the mixture was agitated overnight in a rotary shaker (150 rpm) at room temperature. The mixture was centrifuged at 10,000 g for 10 min, and the supernatant was transferred to a new Ependorf tube. After adding 200 ml of concentrated HCl to the supernatant, the Ependorf tube was transferred to a cold room (4 C) for 4 h to allow the ligninthioglycolic acid (LTGA) derivatives to precipitate. Following centrifugation at 10,000 g for 10 min, the supernatant was discarded, and the orange-brown precipitate was dissolved in 2 ml of 0.5 N NaOH. The absorbance of LTGA derivatives in the supernatant was measured at 280 nm in a spectrophotometer. The concentration of LTGA derivatives was expressed as mg g1 of dried root tissue by using lignin alkali, 2-hydroxypropyl ether (Sigma-Aldrich, USA) as a standard.
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Determination of flavonoid in watermelon roots The methods developed by Pirie and Mullins [35] were used to determine the flavonoid with a few modifications. A 0.5 g sample of watermelon roots was extracted from each replicate with 5 ml of 1% hydrochloric acid-methanol solutions for 24 h in the dark at room temperature. The 0.5 ml of extraction solution was diluted to 10 ml with 1% hydrochloric acid-methanol solution, and the diluted solution was determined spectrophometrically at 325 nm. The flavonoid contents were expressed as mg g1 fresh root tissue by using rutin (Sigma-Aldrich, USA) as a standard.
RNA isolation Total RNA was extracted from the roots and purified using a SK8661 Kit according to the manufacturer's instructions. RNA quality and quantity were determined using a spectrophotometer and agarose gel electrophoresis.
qRT-PCR An AMV First Strand cDNA Synthesis Kit synthesized the firststrand of cDNA using 5 ml of total RNA. This initial strand of cDNA was used as the template for qRT-PCR. The qRT-PCR reactions were run on an ABI StepOne plus real-time PCR system (StepOne, America). Each reaction (20 ml total volume) consisted of 1 ml of eightfold diluted cDNA, 1 ml of primer mix (10 mM of each forward and reverse primer), and 10 ml of SybrGreen qPCR Master Mix (2X). Individual reactions were run with each primer pair at annealing temperatures of 60 C. PCR cycling conditions were as follows: 95 C for 2 min, 40 cycles of 95 C for 10 s, and 60 C for 40 s. Genespecific primers for qRT-PCR were designed based on the EST sequences for six genes, namely: PL (phospholipase), AOS (allene oxide synthase), DAHPS (3-deoxy-D-arabino-heptulosonate-7phosphate synthase), PAL (phenylalanine ammonia-lyase), 4CL (4hydroxycinnamoyl-CoA) and CCOMT (caffeovl-CoA-O-methyltransferase). Table 1 summarizes the specific sets of primers used for the amplification of each cDNA. Melting curves were performed at the end of each reaction run to detect primer dimers and secondary products. The conditions were 95 C for 15 s, followed by 60 C for 1 min, and then kept at 95 C. The watermelon 18S rRNA gene was used as an internal control. The calculation of relative gene expression was conducted as described by Livak and Schmittgen [27]. Each measurement was replicated three times.
Table 1 Genes and primers used in real-time qRT-PCR analysis. Gene
Forward primer
Reverse primer
18S rRNA PL
50 -ATCAGAAAGTAGCA CAACAAGCAC-30 50 -ATTCAGAAATGACGAT GTGGC-30 50 -CGAGAAGGCGAGAGA TGAAGA-30 50 -ATTCGTGATACCTTC CGTGTTC-30 50 -CGAGTTTGGGTTGC CATTTAT-30 50 -CTACATTTTTGGTTGC TCTCTGC-30 50 -GTTTTGGGTGGGTGA TAAGAAG-30
50 -CTTCCGTTCAGCCTTTACCAT-30
AOS DAHPS CCOMT PAL 4CL
50 -TAAGGCGAGCAGATAAGGGAT-30 50 -GCTGAAACACGTGCTATGGTC-30 50 -CTGCCATTCTTCCTACCTTGAT-30 50 -CTCCACCTATCTTCACGAGTTTTTA-30 50 -AAATCTTGAGGGGTGAAGTGTG-30 50 -GGACTATTGATAAAGAAGGATGGC-30
Note: The watermelon 18S rRNA gene was used as an internal control.
Please cite this article in press as: Xu W, et al., Companion cropping with wheat increases resistance to Fusarium wilt in watermelon and the roles of root exudates in watermelon root growth, Physiological and Molecular Plant Pathology (2015), http://dx.doi.org/10.1016/ j.pmpp.2015.02.003
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Assessment of the effects of wheat root exudates on watermelon root growth To assess the effects of wheat root exudates on watermelon root growth, sodium orthovanadate, an inhibitor of root exudates [28], was applied to the gel system [13], consisting of a watermelon seedling planted in agar that had previously contained the D125 wheat as donor plant or without the wheat as control. There were four treatments: 1) D125 (planted D125 wheat in gel system), 2) CK (only planted watermelon), 3) ZD125 (planted D125 wheat and added 30 mmol/L sodium orthovanadate, an inhibitor for root exudate, in gel system) and 4) ZCK (only planted watermelon and added 30 mmol/L sodium orthovanadate in gel system). Triplicates were done for each treatment. D125 wheat seeds were surface sterilized with 0.1% mercuric chloride for 8 min, and rinsed five times with sterile water. The sterilized D125 wheat seeds were planted in petri dish, which contained half-strength Hoagland media, in the dark at 22 C for 48 h to germinate. In D125 and ZD125 treatments, the germinated 25 D125 wheat seeds were transplanted to 250 ml beaker, filled with transparent growth medium consisting of half-strength Hoagland solution and 1.5% phytagel (pH ¼ 5.8). Aluminum foil was used to cover the gel surface and wrap the beaker to keep the roots away from light. Wheat plants were grown at a relative humidity of 70% under a 16/8 h (light/ dark, 22 C/18 C) photoperiod in a growth chamber. The wheat seedlings were pulled out after 7 days growing (the root exudates was still kept in gel system), and then one germinated and sterile watermelon seed was transplanted to beaker. At the same time, one germinated and sterile watermelon seed was transplanted to beaker in CK and ZCK treatments. Watermelon plants were grown at a relative humidity of 70% under a 14/10 h (light/dark, 28 C/ 18 C) photoperiod in a growth chamber. Watermelon roots were harvested on the 30th days after germination, and the harvested watermelon roots were scanned and analyzed root length, root surface area, root volume, root mean diameter, number of roots by root analyzer (LA-S2400) [9].
Fig. 1. Effects of companion cropping with wheat on Fusarium wilt infection in watermelon seedlings. Note: incidence rate of Fusarium wilt in watermelon 15 days after Fon inoculation in sterilized turf soil and vermiculite (1:1, v/v) in growth chamber. Significant differences between treatments were indicated by different letters (P 0.05, independent samples T test). CK: monoculture of watermelon; D125: D125 wheat as companion crop.
accompanied with D125 wheat than in monoculture (Fig. 2). Compared with the monoculture system, companion cropping with wheat increased the activities of PPO by 24.8% in watermelon roots 15 days after Fon inoculation. However, 5 and 10 days after Fon inoculation, no significant difference of PPO activity in the roots of the two cropping systems could be detected (Fig. 2). PAL activity was no difference in watermelon roots for both cropping systems before Fon inoculation. However, PAL activity was higher significantly in watermelon roots of companion cropping than in monoculture after Fon inoculation for 5 days or longer (Fig. 2). Changes of total soluble phenolics, lignin and flavonoid in watermelon roots after exposured to Fon under watermelon-wheat companion cropping
Statistical analyses All data were expressed as mean ± standard error and subjected to ANOVA. The data were analyzed by t-tests method (P 0.05) using the SAS statistical software. Results Responses of companion cropping system to watermelon Fusarium wilt Watermelon plants began to develop symptoms of Fusarium wilt 15 days after inoculation with Fon. However, the incidence rate of watermelon Fusarium wilt was 46.4% in the monoculture system, while the incidence rate of Fusarium wilt in watermelons accompanied with D125 wheat was 13.3% (P < 0.05, independent samples T test), it was significantly lower than monoculture (Fig. 1). Changes of MDA content and enzymatic activities in watermelon roots exposured to Fon under watermelon-wheat companion cropping Before Fon inoculation, MDA (Malondialdehyde) content of the watermelon roots was similar for both the monoculture and the companion cropping with D125 wheat. After Fon inoculation for 5 days, MDA contents started to increase gradually in the roots of watermelon for both cropping systems. However, the increase of MDA content was significantly lower in watermelon roots
Without Fon inoculation, there was no significant difference in total soluble phenolics in watermelon roots for both cropping systems. Total soluble phenolics was higher clearly in watermelon roots of companion cropping than in monoculture at 10 days and 15 days after Fon inoculation (Fig. 2). Before Fon inoculation, the lignin contents were similar in the roots of watermelon for both in monoculture and in companion cropping with D125 wheat. The lignin contents in companion cropping with D125 wheat were significantly higher (22.0%) than in monoculture at 10 days after Fon inoculation. Compared with the monoculture system, lignin content increased up to 21.9% in companion cropping with D125 wheat at 15 days after Fon inoculation (Fig. 2). No significant difference of flavonoid contents could be detected between preinoculation and 10 days post-inoculation. However, flavonoid contents were 54.2% higher in D125 companion system than in monoculture at 15 days after Fon inoculation (Fig. 2). Responses of gene expressions involved in JA biosynthesis and shikimate-phenylpropanoid-ligin pathways to Fon This study analyzed the expression of genes in JA biosynthesis and shikimate-phenylpropanoid-lignin pathways, which were related in the watermelon roots under companion cropping and monoculture systems after challenging by Fon [29]. Phospholipase (PL) and allene oxide synthase (AOS) are involved in the different steps of the JA biosynthesis pathway. Results from qRT-PCR showed that the expressions of PL and AOS were significantly increased at 4,
Please cite this article in press as: Xu W, et al., Companion cropping with wheat increases resistance to Fusarium wilt in watermelon and the roles of root exudates in watermelon root growth, Physiological and Molecular Plant Pathology (2015), http://dx.doi.org/10.1016/ j.pmpp.2015.02.003
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Fig. 2. Responses of MDA content, PAL activity, PPO activity, flavonoid content, total soluble phenolics and lignin content in watermelon roots to the presence of wheat as companion crop and to infection with Fon. MDA: Malondialdehyde; PAL: Phenylalanine ammonia-lyase; PPO: Polyphenoloxidase; CK: monoculture of watermelon; D125: D125 wheat as companion crop; Pre-: before Fon inoculation; Post-5 d: 5 days after Fon inoculation; Post-10 d: 10 days after Fon inoculation; Post-15 d: 15 days after Fon inoculation.
8 and 12 h after Fon inoculation in the companion cropping system. However, in the monoculture system, expressions of PL and AOS genes were increased slightly at 24 h after Fon inoculation (Fig. 3). Expressions of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS), phenylalanine ammonia-lyase (PAL), 4-hydroxycinnamoyo-CoA ligase (4CL), and caffeovl-CoA O-methyltransferase (CCOMT), which involved in different steps of the shikimatephenylpropanoid-lignin biosynthesis pathway, were also analyzed. DAHPS expression was upregulated at 1 and 5 days after inoculation in the companion cropping system, whereas its expression was upregulated at 3 and 8 days after inoculation in
monoculture. PAL expression level was higher at 1 and 8 days after inoculation in companion cropping than in monoculture system. 4CL expression was upregulated at 1 and 8 days after inoculation in the companion cropping system, whereas its expression level was lower at 3 and 5 days after inoculation in the companion cropping system than in monoculture system. CCOMT expression was induced at 1, 3, 5 and 8 days after inoculation in companion cropping whereas CCOMT expression was similar to the preinoculation levels at these time point in the monoculture (Fig. 4). Effect of D125 wheat root exudates on root growth of watermelon seedlings The root length, root surface area, root volume, root dry weight and root numbers of watermelon seedlings in the D125 treatment in the gel system (D125) were higher than those in the control (CK), whereas root mean diameter for watermelon seedlings was thinner compared to CK (Fig. 5). Interestingly, the effect was abolished while sodium orthovanadate added in gel system, there were no significant differences on root length, root surface area, root volume, root number, root mean diameter and root dry weight in watermelon seedlings in ZD125 and ZCK (Fig. 6). Discussion
Fig. 3. Expressions of PL and AOS genes involved in JA biosynthesis in watermelon when grown alone (CK) or together with wheat variety D125 (D125) before and after inoculation with Fon. Results from qRT-PCR. The JA biosynthetic pathway was adopted from Creelman and Mullet [10]. PL: phospholipase; AOS: allene oxide synthase; Pre-: before Fon inoculation; Post-4 h: 4 h after Fon inoculation; Post-8 h: 8 h after Fon inoculation; Post-12 h: 12 h after Fon inoculation; Post-24 h: 24 h after Fon inoculation.
Some findings showed the importance of root exudates in defending the plant against pathogenic microorganisms [38,51]. Hao et al. [19] demonstrated the mechanism in which rice root exudates inhibited spore germination and sporulation of Fon in a watermelon/aerobic rice intercropping system. In previous study, we found that there was no significant difference in mycelium growth of Fon with or without the D125 wheat root exudates [48]. However, this experiment showed that watermelon seedlings could resist against Fusarium wilt in the companion cropping with D125 wheat system as compared to monoculture (Fig. 1). The reasons
Please cite this article in press as: Xu W, et al., Companion cropping with wheat increases resistance to Fusarium wilt in watermelon and the roles of root exudates in watermelon root growth, Physiological and Molecular Plant Pathology (2015), http://dx.doi.org/10.1016/ j.pmpp.2015.02.003
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Fig. 4. Expressions of some genes involved in shikimate-phenylpropanoid-lignin biosynthesis in companion cropping and monoculture interactions between watermelon and Fon via qRT-PCR. The shikimate-phenylpropanoid-lignin biosynthetic pathway was adopted from Whetten and Sederoff [43]. DAHPS: 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase; PAL: phenylalanine ammonia-lyase; 4CL: 4-hydroxycinnamoyl-CoA ligase; CCOMT: caffeovl-CoA-O-methyltransferase. Pre-: before Fon inoculation; Post-1 d: 1 d after Fon inoculation; Post-3 d: 3 d after Fon inoculation; Post-5 d: 5 d after Fon inoculation; Post-8 d: 8 d after Fon inoculation.
may be that D125 wheat root exudates promoted the growth of watermelon roots (Fig. 5). When plants are attacked by pathogens or placed under some other stress, a series of physiological and biochemical responses could occur in the plants [8,15]. MDA content represents the degree of damage to plant cell membranes by the stress period, the higher the level of MDA is, the more severe the damage to the cell [32]. This experiment showed that companion cropping with D125 wheat decreased MDA content in watermelon roots after inoculation with Fon compared with monoculture (Fig. 2). This change of content in MDA implied that companion cropping with D125 wheat increased
the stability of biological membranes to prevent infection of watermelon from Fon. In this study, companion cropping with D125 wheat significantly increased the PPO activities in watermelon roots compared with monoculture (Fig. 2). Due to the increase of PPO activity the more lignins and other phenolics were synthesized to make cell walls stronger, so that it prevented invasion from pathogen [23,37]. Hassan et al. [20] reported that the resistance against bacterial wilt of potato was induced by application of plant extracts, which was associated with the increase of PAL activities. In our investigation, PAL activity in watermelon roots was higher at 5, 10, 15 days after
Fig. 5. The effect of wheat root exudates on root length, root surface area, root volume, root mean diameter, number of roots, root dry weight in watermelon seedlings. CK: only planted watermelon in gel system where was not added sodium orthovanadate; D125: D125 wheat were planted in gel system where was not added sodium orthovanadate, wheat seedlings were pulled out after 7 days growing, and then a watermelon seedling was planted in gel system.
Please cite this article in press as: Xu W, et al., Companion cropping with wheat increases resistance to Fusarium wilt in watermelon and the roles of root exudates in watermelon root growth, Physiological and Molecular Plant Pathology (2015), http://dx.doi.org/10.1016/ j.pmpp.2015.02.003
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Fig. 6. The effect of wheat root exudates on root length, root surface area, root volume, number of roots, root mean diameter, root dry weight in watermelon seedlings after adding root exudates in the gel system. ZCK: only planted watermelon in gel system where was added sodium orthovanadate; ZD125: D125 wheat were planted in gel system where was added sodium orthovanadate, wheat seedlings were pulled out after 7 days growing, and then a watermelon seedling was planted in gel system.
Fon inoculation in companion cropping with D125 wheat compared with monoculture. This implied that D125 wheat root exudates could induce resistance against Fusarium wilt in watermelon and delay the occurrence of Fusarium wilt in watermelon. Nicholson and Hammerschmidt [34] reported that phenolic compounds formed in response to infection with the pathogen were considered as part of an active defense response and consequently protected plants against pathogenic attacks. Phenolic compounds could play a major role in wilt disease resistance through the inactivation of fungal cell wall degrading enzymes (cellulase and pectin methyl esterase), thereby restricting the degradation of the cell wall and the fungal invasion in plants [30]. In the present work, there was a noticeable increase in total phenolic compounds in watermelon roots at 10 and 15 days after Fon inoculation in companion cropping with D125 wheat compared with monoculture (Fig. 2). It is suggested that companion with D125 wheat enhanced resistance in watermelon to Fusarium wilt by increasing total phenolic compounds in watermelon roots. Flavonoids are a broad group of secondary metabolites and have been reported to confer disease resistance in different plant species [3]. Lignin, a major component of cell walls of vascular plants, was considered as a first line defense against penetration of invasive pathogens [6]. At 15 days after Fon inoculation, flavonoid content in watermelon roots were higher in companion cropping with D125 wheat than in monoculture (Fig. 2). At 10 and 15 days after Fon inoculation, lignin content was higher in watermelon roots in companion cropping with D125 wheat than in monoculture. It implied that companion with D125 wheat prevented invasion of pathogens by increasing the content of secondary metabolism. Interestingly, the content of MDA became lower in companion cropping system at 5 days after Fon inoculation, compared to the control. It suggested that the damage of membrane was less in companion cropping system than in monoculture. Thus, increasing of the membranes stability was a key factor for reducing Fusarium wilt in watermelon in companion cropping system. The compounds generated from phenylpropanoid and jasmonate pathways play the major roles in plant defense responses against pathogen attack [12,39]. Lü et al. [29] demonstrated that JA
biosynthesis and shikimate-phenylpropanoid-lignin pathways are important processes in watermelon resistance against Fon infection. We found in this study, expression levels of the genes in responsible for biosynthesis of JA and shikimate-phenylpropanoidlignin were enhanced in both monoculture and companion cropping with Fon inoculation. However, the timing and levels of the expressions were different between the two cropping system. In the companion cropping system, PL and AOS gene expression were strongly enhanced at 4 h, 8 h and 12 h after Fon inoculation and the levels were higher than in the monoculture system (Fig. 3). The expression level of DAHPS gene was higher in companion cropping than in monoculture at 1 d and 5 d after Fon inoculation. The expression levels of PAL and 4CL gene were higher in companion cropping than in monoculture at 1 d and 8 d after Fon infection, also the CCOMT gene expression level was strongly upregulated in companion cropping at 1, 3, 5 and 8 days after Fon inoculation (Fig. 4). Our results showed that the timing points for the gene expression were earlier than the other indicators, and the response was stronger in companion cropping with D125 wheat than in monoculture. Therefore, we speculate that the resistance against Fon was increased in companion cropping system by the high expression of the six genes after inoculated with Fon. In the previous study, we found that companion cropping wheat promoted growth of watermelon [49,50]. The phytochemicals presented in the root exudates of plants mediated several types of communication processes in the rhizosphere such as rooteroot, root-microbe and rooteinsect interactions [4,41]. Loyala-Vargas et al. [28] demonstrated that the exudation of some compounds in Arabidopsis root was inhibited by sodium orthovanadate. In this study, we compared treatments with and without the addition of sodium orthovanadate to the gel system to evaluate the role of wheat root exudates in promoting watermelon root growth. It was observed that D125 wheat root exudates have significant positive effects on the growth of watermelon roots (Fig. 5). When sodium orthovanadate was added in the gel system, D125 wheat root exudates (ZD125) had no effect on the growth of watermelon roots, which suggests that the exudation of some compounds in D125 wheat root were inhibited (Fig. 6). This study indicated that D125
Please cite this article in press as: Xu W, et al., Companion cropping with wheat increases resistance to Fusarium wilt in watermelon and the roles of root exudates in watermelon root growth, Physiological and Molecular Plant Pathology (2015), http://dx.doi.org/10.1016/ j.pmpp.2015.02.003
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wheat root exudates had an important role in promoting the growth of watermelon roots. These results support the role of root exudates in the phenomenon that companion cropping with D125 wheat could increase growth of watermelon. Caffaro et al. [7] evaluated the role of secondary metabolites in the development of root architecture by comparison of treatments between without and with the addition of activated charcoal to cropping systems, and indicated that secondary metabolites presented in the root exudates played key roles on the development of root architecture. Logically, natural plant growth regulators may be involved in root facilitation interactions [21], even if we did not identify what they are still. Further studies are needed to define the functional components in D125 wheat root exudates that promote the growth of watermelon root. In the conclusion, companion cropping with D125 wheat could reduce disease incidence of Fusarium wilt in watermelon by promoting growth of watermelon roots and inducing gene expression and physiological changes to protect watermelon from the infection. Further research is needed to identify the functional components in D125 wheat root exudates that promote watermelon roots growth, and also it is needed to investigate the effects of companion cropping with D125 wheat on the microbial community diversity in the rhizosphere soil with watermelons in the field experiments. Acknowledgments We gratefully acknowledge National Nature Science Foundation of China (31471917) and Science and Technology Research Project of Heilongjiang Province Department of Education, China (12541859) for theirs funding of this project. References [1] An MJ, Wu FZ, Liu B. Study on the differentiation of physiological race from Fusarium oxysporum f.sp.niveum and the resistance of different watermelon cultivars in Heilongjiang. J Shanghai Jiaot Univ 2009;27(5):494e500 [in China]. [2] Badri DV, Vivanco JM. Regulation and function of root exudates. Plant Cell Environ 2009;32:666e81. [3] Bahraminejad S, Asenstorfer RE, Riley IT, Schutz CJ. Analysis of antimicrobial activity of flavonoids and saponins isolated from the shoots of oats (Avena sativa L.). J Phytopathol 2008;156:1e7. [4] Bais HP, Park SW, Weir T, Callaway RM, Vivanco JM. How plants communicate using the underground information superhighway. Trends Plant Sci 2004;9: 26e32. [5] Barber MS, Ride JP. A quantitative assay for induced lignifications in wounded wheat leaves and its use to survey potential elicitors of the response. Physiol Mol Plant P 1988;32(2):185e97. [6] Bhuiyan NH, Selvaraj G, Wei YD, King J. Role of lignifications in plant defense. Plant Signal Behav 2009;4(2):158e9. [7] Caffaro MM, Vivanco JM, Gutierrez Boem FH, Rubio G. The effect of root exudates on root architecture in Arabidopsis thaliana. Plant Growth Regul 2011;64:241e9. langer RR, Benhamou N, Paulitz TC. Defense enzymes induced in [8] Chen C, Be cucumber roots by treatment with plant growth-promoting rhizobacteria (PGPR) and Pythium aphanidermatum. Physiol Mol Plant P 2000;56:13e23. [9] Chu LR, Pan K, Wu FZ, Tao L, Wang Y. Effects of Hexadecanoic acid on Fusarium oxysporum f. sp. niveum control and on growth of watermelon. Allelopathy J 2014;34(2):241e52. [10] Creelman RA, Mullet JE. Jasmonic acid distribution and action in plants: Regulation during development and response to biotic and abiotic stress. Proc Natl Acad Sci U S A 1995;92:4114e9. [11] De-la-Pena C, Lei Z, Watson BS, Sumner LW, Vivanco JM. Root-microbe communication through protein secretion. J Biol Chem 2008;283:25247e55. [12] Dixon RA, Paiva NL. Stress-induced phenylpropanoid metabolism. Plant Cell 1995;7:1085e97. [13] Fang SQ, Gao X, Deng Y, Chen XP, Liao H. Crop root behavior coordinates phosphorus status and neighbors: from field studies to three-dimensional in situ reconstruction of root system architecture. Plant Physiol 2011;155: 1277e85. [14] Fravel DR, Deahl KL, Stommel JR. Compatibility of the biocontrol fungus Fusarium oxysporum strain CS-20 with selected fungicides. Biol Control 2005;34:165e9. s JA, Jime nez-Díaz RM, Tena M. In[15] Garcia-Limones C, Herv as A, Navas-Corte duction of an antioxidant enzyme system and other oxidative stress markers
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