Eur J Plant Pathol (2014) 140:643–653 DOI 10.1007/s10658-014-0494-6
Effects of Fusarium oxysporum on rhizosphere microbial communities of two cucumber genotypes with contrasting Fusarium wilt resistance under hydroponic condition Ju Ding & Yiqing Zhang & Huan Zhang & Xin Li & Zenghui Sun & Yangwenke Liao & Xiaojian Xia & Yanhong Zhou & Kai Shi & Jingquan Yu Accepted: 31 July 2014 / Published online: 5 August 2014 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2014
Abstract Two cucumber genotypes were chosen that differ in Fusarium wilt resistance and inoculated with Fusarium oxysporum (Schlechtend, Fr) f. sp. cucumerinum (Owen) Snyder & Hansen (FO) in a hydroponic nutrient solution system to determine whether differences in plant genotype affected rhizosphere microbial community and its involvement in resistance to root-borne pathogens. Response of the FO population and microbial communities were compared in the nutrient solution and on root surfaces. Results demonstrated that FO inoculation resulted in a higher FO population on root surfaces and a lower population in the nutrient solution for the susceptible genotype JinYan NO. 4 (JYan); however, an inverse pattern was observed in the resistant JinYou NO. 1 (JYou). Similarly, promotive effects on FO spore germination and germ-tube J. Ding : Y. Zhang : H. Zhang : X. Li : Z. Sun : Y. Liao : X. Xia : Y. Zhou : K. Shi (*) : J. Yu Department of Horticulture, Zijingang Campus, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, People’s Republic of China e-mail:
[email protected] J. Ding Seed Management Station of Xiaoshan District, 1169 Gongxiu Road, Hangzhou 311201, People’s Republic of China J. Yu Key Laboratory of Horticultural Plants Growth, Development and Quality Improvement, Agricultural Ministry of China, 866 Yuhangtang Road, Hangzhou 310058, People’s Republic of China
elongation were only observed in root exudates from FO-inoculated susceptible JYan. FO inoculation resulted in overall increases in cultivable fungi and oomycetes, and actinomycete populations on root surfaces of the resistant JYou compared to the JYan counterpart. PCR-DGGE analysis of 16S rDNA fragments indicated that following FO inoculation, significant changes in the bacterial structure isolated from the root surfaces of susceptible plants were observed, such as an increased diversity index, a decreased evenness index, and the occurrence of several types of bacteria that decomposed organic substances. These results suggest that a complex interaction between plant genotypes and pathogen affected the rhizosphere microbial community. Keywords Microbial community . Root exudates . Cucumis sativus . Fusarium oxysporum . Resistance . Hydroponic system
Introduction The productivity and health of agricultural soil systems depend greatly on the functional processes of soil/rhizosphere microorganisms and the microbial community. Plant-associated rhizosphere bacteria improve plant growth and development by different mechanisms including nitrogen fixation, improvement of nutrient uptake, synthesis of growth-promoting substances, and protection of plants against phytopathogens (Arif et al. 2013; Dardanelli et al. 2012). Several plant
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growth-promoting rhizobacteria (PGPR) and pathogensuppressive microorganisms have been isolated from soils (Nico et al. 2012). After plant contact with a number of beneficial bacteria, such as Pseudomonas fluorescens and P. putida, induced systemic resistance (ISR) can occur (Schuhegger et al. 2006; Zamioudis and Pieterse 2012). Disease suppression is observed worldwide in soil and soilless systems, and it is reported to be highly correlated with the cultivable number of filamentous actinomycete and bacterial composition (Postma et al. 2005). Microbial activity is enhanced in the rhizosphere, there is evidence that the interaction between plants and microbes is bi-directional (Micallef et al. 2009). Abundant studies revealed that microbial communities in the rhizosphere are primarily plant-driven, responding to density, composition, abundance and diversity of plant-derived exudates (Mitter et al. 2013). The plant root system acts as a chemical factory and exudes enormous amounts of chemicals to effectively communicate with the surrounding soil organisms (Mitter et al. 2013). Apart from being a major source of energy and nutrients for the microbes in the rhizosphere, molecules exuded by the plant may act as signals which influence the ability of the microbe to colonize the host or survive in the rhizosphere (Mark et al. 2005; Shidore et al. 2012). Root exudates also play a critical role in plant resistance to diseases. Plants secrete phytoalexins, defence proteins, and other unknown chemicals, in response to pathogenic microorganisms’ infection (Flores et al. 1999; Nóbrega et al. 2005). Root exudates involve secretion of antimicrobials, nematicidal, and insecticidal compounds, such as indole, terpenoid, benzoxazinone, flavonoid/isoflavonoid, rosmarinic acid, and pigmented naphthoquinones have been identified (Bais et al. 2006; Hartmann et al. 2009). These findings strongly suggest the importance of root exudates in defending the rhizosphere against pathogenic microorganisms. Plant specific effects are associated with differences in the composition of root cells and root exudates (Kamilova et al. 2006; Tian et al. 2013). An emerging body of evidence has indicated a role for plant species as a determinant factor in the genetic composition of the rhizosphere microbial community (Li et al. 2011). A previous study demonstrated that plants, such as oilseed rape and strawberry, have host-specific rhizosphere bacterial communities (Costa et al. 2006). These plantmicrobe interactions are far from confined to plant
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species; eight Arabidopsis accessions were found to exert a marked selective influence on bacteria associated with their roots (Micallef et al. 2009). Many plant genotypes with resistance to soil-borne pathogens have been developed. However, little information is available on the relationship between soil-borne pathogens and the microbial community after expose to genotypes with different resistance to soil-borne pathogens. An understanding of such relationships is vital for the management of agricultural ecosystems under pathogenchallenging conditions. Cucumber (Cucumis sativus L.) is a major greenhouse vegetable worldwide, and Fusarium wilt, caused by Fusarium oxysporum (Schlechtend, Fr) f. sp. cucumerinum (Owen) Snyder & Hansen (FO), is a root pathogen that causes serious losses in cucumber production (Ye et al. 2004). We hypothesized that rhizosphere microbial community was involved in the plant genotypic difference in resistance to root-borne pathogens. Two cucumber genotypes with contrasting resistance to Fusarium wilt were cultivated hydroponically. The occurrence of Fusarium wilt, fungal activity in root exudates, FO pathogen populations, microorganism populations, and bacterial community structures on root surfaces were investigated. Conventional cultural methods were used to monitor pathogen levels and microbial populations, and 16S rDNA gene profiling was used to investigate the bacterial community. Cultural methods detected major perturbations in microbial populations, and the molecular approach provided greater sensitivity and insight into the community structure.
Materials and methods Greenhouse experiments Two cucumber genotypes (Cucumis sativus L. c.v. JinYou No. 1 and JinYan No. 4) were used in this experiment. JinYou No. 1 (JYou) is resistant to the pathogen Fusarium oxysporum, and JinYan No. 4 (JYan) is susceptible. Seeds, obtained from the Zhejiang Academy of Agricultural Sciences, China, were germinated in trays of growth medium containing a mixture of vermiculite and perlite (1/1, v/v) in a greenhouse. After emergence, batches of eight seedlings were transferred to plastic tanks (13 L) filled with 10 l of half-strength Enshi nutrient solution (prepared with tap water) for hydroponic culture (Yu and Matsui 1994).
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Seedlings were supported by floating polystyrene foam, rootstock of each seedling was wrapped with sponge strips and placed into one of punched small holes to keep the seedlings upright. Air temperature was maintained at 28/16 °C (day/night), and relative humidity ranged between 80 and 95 %. Half of the plants from each genotype were inoculated with FO via the addition of a conidial suspension into the nutrient solution when the cucumber seedlings were at the two-leaf stage. FO used in this study was Fusarium oxysporum (Schlechtend, Fr) f. sp. cucumerinum (Owen) Snyder & Hansen (provided by Mie Agricultural Experiment Station, Japan). FO isolate was incubated in liquid potato sucrose medium with shaking (200 rpm) at 28 °C for 6 days. The microconidia-like budding cells of FO were filtered with two layers of cheese cloth to remove filamentous mycelia, harvested by centrifugation (2,000 × g, 10 min), and then washed twice with sterile distilled water. The harvested cells were suspended and added to the nutrient solution to obtain a final concentration of 1.0×104 conidia ml−1 (Yu and Komada 1999). The resulting four treatments were designated JYou, JYou+ FO, JYan, and JYan+FO. The experiment was terminated 25 days post-inoculation when FO-inoculated JYan plants displayed wilting, yellowing or death. Measurement of Fusarium wilt and the FO population The percentage of leaf yellowing or wilting of plants and FO populations were measured 25 days post-inoculation. Each plant was harvested for measurements of root rot and vascular bundle browning on a scale of 0–4: 0, healthy without any browning; 1, white root with scarce browning (under 5 % of total root); 2, light root rot and browning (5–20 % of total root); 3, mild root rot and browning (20–50 % of total root); and 4, severe root rot and browning (50–100 % of total root) (Ye et al. 2004). The rhizosphere microflora was collected using a method similar to that described by Khalil and Alsanius (2001). Five grams of fresh roots from each treatment was added to 20 ml of sterile distilled water and shaken at 60 rpm for 3 h, and the resulting suspension, which presumably contained the microbial community from the root surfaces, was collected. The prepared suspensions and the hydroponic nutrient solutions from the tanks were diluted with sterilized water and plated onto a semiselective Komada medium (Komada 1975). The number of colony-forming units (CFUs) was
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counted after 6 days at 28 °C, and the CFU of FO on root surfaces and in nutrient solutions was estimated accordingly. Microbial population analysis The solutions prepared from roots were also used for the analysis of microorganism populations on root surfaces. Diluted solutions were plated onto beef extract-peptone agar medium, Martin’s rose-bengal agar medium, and Gaoshi No. 1 agar medium for the enumeration of bacterial, fungal and oomycete, and actinomycete populations, respectively (Wollum 1982). After 2 d at 37 °C for bacteria, 4 d at 28 °C for fungi and oomycetes, 4 d at 28 °C for actinomycetes, respectively, the number of CFU was counted and the number of viable cells was estimated. Fungal activity in the root exudates The percentage of spore germination and germ-tube length were determined via the incubation of FO spores in exudates from the roots of JYou and JYan, with or without FO inoculation, to determine the genotypic effects of root exudates on fungal activity (He et al. 2002). After 2 days of FO inoculation in the above mentioned hydroponic system, root exudates were collected by submerging the root system (two plants at twoleaf stage) into 30 ml of sterile distilled water in 100 ml conical flasks in darkness for 24 h. The resulting solutions were collected and sterilized using filtration at 0.22 μm and stored at 4 °C before use within 12 h. FO spores were harvested from 2-week-old cultures, grown on potato dextrose agar, by dislodging of spores with a glass rod into distilled water. Spore suspensions were filtered through cheesecloth to remove mycelial fragments. The filtrate was centrifuged at 5,000×g for 10 min, and the spore pellet was resuspended in distilled water, quantified, and adjusted to 1×106 spores ml−1. Two millilitres of the spore suspension was diluted with 2 ml of root exudate solution or sterile distilled water and incubated at 28 °C. At least 150 spores per treatment were examined for the percentage of spore germination and germ-tube length after 48 and 72 h, respectively, using a light microscopy equipped with a digital camera (Leica Microsystems, Wetzlar, Germany), and analyzed using the image analysis software Adobe Photoshop CS5 (Adobe, San Jose, CA, USA).
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DNA extraction and community fingerprinting by PCR-DGGE Root surface microbial DNA was extracted using a commercial genomic DNA isolation Kit (Sangon, Shanghai, China). Solutions were prepared as described above for enumeration of microbial population on root surfaces. The quality and quantity of extracted DNA were confirmed using electrophoresis through a 1 % agarose gel. DNA PCR was performed using a GeneAmpTM PCR System 9700 (Applied Biosystems) with the following primers for the bacterial groups: 341fGC (5′-GC Clamp [CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G]-CCT ACG GGA GGC AGC AG-3′) and 534r (5′-ATT ACC GCG GCT GCT GG-3′) (Muyzer et al. 1993). Each PCR reaction mixture consisted of 6 μl 10× PCR buffer, 0.6 μl 10 mM deoxynucleoside triphosphate mixture, 1 μl of each 10 μM primer, 0.6 μl Taq DNA polymerase (Sangon, Shanghai, China), 1 μl template DNA (5–15 ng), and sterile distilled water to supplement the reaction mixture to a total volume of 60 μl. PCR amplification was performed at 94 °C for 5 min, followed by 35 thermal cycles of 94 °C for 30 s, 54 °C for 30 s, 72 °C for 30 s, and a final single extension at 72 °C for 5 min. The size of the PCR product was visualized using electrophoresis in 1 % agarose gels after ethidium bromide (EB) staining. Strong bands of approximately 230 bp were subjected to denaturing gradient gel electrophoresis (DGGE) analysis. DGGE was performed using a DcodeTM Universal Mutation Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Fifty microliters of PCR product of each sample was loaded onto 10 % polyacrylamide gels in 1× TAE (9 mM Tris and 2 mM EDTA). Polyacrylamide gels with a 30–60 % denaturant gradient (100 % denaturant contains 7 M urea and 40 % deionized formamide) were run at 180 V and 60 °C for 270 min. The gels were stained with EB for 20 min and photographed under UV light using Tanon equipment (Tanon Technology, Shanghai, China). Photographs were analyzed using a Tanon GIS gel photograph management system (Tanon Technology, Shanghai, China). Structural diversity from DGGE data was evaluated. The Shannon-Weaver diversity index (H) was calculated as follows: H=−Σ(pi)(log2 pi), where pi is the proportion of ith phylotype (Shannon and Weaver 1963). The Simpson’s diversity index (D) was calculated using
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the formula D=1-Σ(pi)2, and the results are reported as the reciprocal (1/D) (Simpson 1949). Evenness (E) was calculated from H/ln(S), where S is the total number of phylotypes (Shannon and Weaver 1963). Gel strips of bands were excised from the DGGE gel into a 1.5 mL tube, and DNA was eluted in 50 μl of TE (pH 8.0) at 37 °C for 2 h. The DNA fragments were amplified from the eluted solution using PCR. The primer pair without the GC clamp (341f and 534r) was used in the PCR template amplification for sequencing. PCR products were cloned into pGEM-T easy vectors (Promega, USA), and sequencing was conducted as described by Green et al. (2004). Sequences of DNA recovered from excised bands were submitted to the National Center for Biotechnology Information (NCBI) for BLAST analysis. Bacterial classification was determined using a sequence match program (Ribosomal Database Project II-Release 9 website). Statistical analysis There were six hydroponic tanks (eight plants per tank) for each treatment. Nutrient solution samples for FO population were collected randomly from three tanks for each treatment. Sampling from root surface FO populations, microorganism populations and PCRDGGE analysis were from at least three plants from different tanks. Sampling for root exudate fungal activities were collected from six plants (two plants per replicates, and three replicates for every treatment). At the end of the experiment, each plant was harvested for determination of Fusarium wilt incidence and the browning index of the vascular bundle. The data analysis for this paper was generated using SAS software, Version 8 of the SAS System for Windows. Copyright © 1999– 2000 SAS Institute Inc. SAS and all other SAS Institute Inc. product or service names are registered trademarks or trademarks of SAS Institute Inc., Cary, NC, USA. The mean values were compared using Duncan’s multiple range test (P
