Plant Physiology and Biochemistry 83 (2014) 250e257
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Quantification and role of organic acids in cucumber root exudates in Trichoderma harzianum T-E5 colonization Fengge Zhang a, Xiaohui Meng a, Xingming Yang a, b, Wei Ran a, b, *, Qirong Shen a, b a b
National Engineering Research Center for Organic-based Fertilizers, Nanjing Agricultural University, Nanjing 210095, PR China Jiangsu Collaborative Innovation Center for Solid Organic Waste Utilization, Nanjing Agricultural University, Nanjing 210095, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 April 2014 Accepted 14 August 2014 Available online 23 August 2014
The ability to colonize on plant roots is recognized as one of the most important characteristics of the beneficial fungi Trichoderma spp. The aim of this study is to prove that the utilization of organic acids is a major trait of Trichoderma harzianum T-E5 for colonization of cucumber roots. A series experiments in split-root hydroponic system and in vitro were designed to demonstrate the association between the utilization of organic acids and T-E5 colonization on cucumber roots. In the split-root hydroponic system, inoculation with T-E5 (T) significantly increased the biomass of cucumber plants compared with CK (non-inoculation with T-E5). The T-E5 hyphae densely covering the cucumber root surface were observed by scanning electron microscopy (SEM). Three organic acids (oxalic acid, malic acid and citric acid) were identified from both the CK and T treatments by HPLC and LC/ESI-MS procedures. The amounts of oxalic acid and malic acid in T were significantly higher than those in CK. All the organic acids exhibited different and significant stimulation effects on the mycelial growth and conidial germination of T-E5 in vitro. An additional hydroponic experiment demonstrated the positive effects of organic acids on the T-E5 colonization of cucumber roots. In conclusion, the present study revealed that certain organic acids could be used as nutritional sources for Trichoderma harzianum T-E5 to reinforce its population on cucumber roots. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Colonization HPLC Nutritional source Organic acids Split-root hydroponic system
1. Introduction Trichoderma spp., as well-known rhizosphere-resident antagonistic fungus, is widely used to control plant diseases caused by numerous soil-borne pathogens, such as Rhizoctonia solani (Huang et al., 2011), Rhizopus oryzae (Howell, 2002), Fusarium spp. (Zhang et al., 2013a), etc. Moreover, Trichoderma strains have the ability to promote plant growth and development by increasing plant nutrient uptake, photosynthetic and respiratory efficiency, and production of growth hormones (Contreras-Cornejo et al., 2009; Zhang et al., 2013b). Many reports also show that the combination of organic fertilizers and Trichoderma strains is a good way to control plant diseases or facilitate high-yield crops (Chen et al.,
Abbreviations: CFU, colony forming units; HPLC, high performance liquid chromatography; LC/ESI-MS, liquid chromatography/ electrospray ionization-mass spectrometry; PDA, potato dextrose agar; SEM, scanning electron microscopy; UV, ultra violet. * Corresponding author. Postal address: College of Resources and Environmental Sciences, Nanjing Agricultural University, 210095 Nanjing, Jiangsu Province, PR China. Tel.: þ86 025 84396212; fax: þ86 025 84396824. E-mail address: [email protected] (W. Ran). http://dx.doi.org/10.1016/j.plaphy.2014.08.011 0981-9428/© 2014 Elsevier Masson SAS. All rights reserved.
2012; Zhang et al., 2013c). Evidence has accumulated indicating that some Trichoderma strains have good colonization abilities (Rigot and Matsumura, 2002; Brotman et al., 2013) and can successfully survive on root surfaces and even on entire roots (Bailey et al., 2006; Bae et al., 2009). In practice, the survival rate and population density of beneficial microorganisms are the prerequisites for their effectiveness or specific functions in the rhizosphere (Raaijmakers et al., 2009). Moreover, the quality and effectiveness of biological agents are also determined by the continuous extension abilities of functional strains during the €m colonization of soil or plant roots in the field. For instance, Alstro € m, 2000) demonstrated that the biocontrol effect of Tricho(Alstro derma was directly dependent on its rhizosphere colonization ability, and Trichoderma proved able to colonize lettuce roots to successfully control the disease caused by Sclerotina minor in field experiments (Rabeendran et al., 2006). The rhizosphere is a dynamic system in which a number of interactions and interdependent relationships between plant roots and microorganisms take place (Mogan et al., 2005). Moreover, as the medium of roots and surrounding microorganisms, root exudates play an important role in information transmission between them. Different plants have different root exudates, and the root
F. Zhang et al. / Plant Physiology and Biochemistry 83 (2014) 250e257
exudates released to the rhizosphere during the process of plant growth include a wide range of organic and inorganic materials (Badri and Vivanco, 2009). The composition and quantity of these compounds directly affect the dominant species and breeding of microorganisms in the rhizosphere (Zhang et al., 2013a). The root exudates, such as sugars, amino acids, phenolic acids and organic acids (Swarnalee et al., 2013), have significant stimulatory or inhibitory effects on the growth of microorganisms (Hao et al., 2010; Ling et al., 2011a) and can be used as nutrients or signals by some beneficial microorganisms (Randy et al., 2009). The rhizosphere microorganisms are able to use soluble root exudates compounds as their major carbon nutrient source, which is considered to be the nutritional basis to guarantee successful rhizosphere colonization (Hiltner, 1904). Raaijmakers et al. (2009) also demonstrated that these root exudates are the main food sources for microbes and a driving force of their population density and activities. Low-molecular-weight organic acids are usually present in root exudates, of which they are reported as a main component (Liu and Wen, 2006). Many studies have focused on demonstrating the functions of organic acids involved in the metabolic processes of pez-Bucio et al., 2000; Lilia et al., 2011) and shown the plants (Lo chemotaxis response of bacteria toward different organic acids (Tan et al., 2013). However, few studies have investigated the effects of organic acids on the activities of rhizosphere fungi (for example, Trichoderma spp.) concerning the relationship between organic acids and colonization ability of fungi in the rhizosphere. In this study, we hypothesized that organic acids could be used as a nutritional basis for rhizosphere fungi (Trichoderma spp.) to support their colonization of cucumber roots. Therefore, our study aimed to investigate the colonization of Trichoderma harzianum TE5 hyphae on cucumber roots using scanning electron microscope (SEM) in a split-root hydroponic system; to identify and measure the organic acids in cucumber root exudates; and to confirm the role of organic acids (used as nutritional sources to attract T. harzianum) in T-E5 colonization of cucumber roots. 2. Methods 2.1. Microorganisms and microconidia Trichoderma harzianum strain T-E5 (CCTCC No.AF2012011, China Center for Type Culture Collection) was provided by Jiangsu Provincial Key Lab of Organic Solid Waste Utilization, Nanjing, China and used throughout this study. The strain was routinely incubated on potato dextrose agar (PDA) medium in Petri dishes in the dark at 28 C for 7 days and maintained at 80 C in 30% glycerol for longterm storage. The conidial suspension of Trichoderma harzianum TE5 was prepared according to Zhang et al. (2013a). The conidia concentration was 1.8 107 CFU mL1, which was determined by direct observation on a hemacytometer (Hinotek Technology, Zhongguancun, China).
251
leaf unfolded, which roughly took 7 days. Seedlings at a similar stage were carefully removed from the disk, and the roots were lightly washed 3 times with sterilized double-distilled water before they were moved into the split-root hydroponic system. 2.3. Description of the split-root hydroponic experiment A split-root hydroponic experiment was designed mainly for the followed cucumber root exudates experiment in this study. The split-root hydroponic system was employed according to Ling et al. (2011b), with a few modifications. The system consisted of two compartments (Fig. 1A, compartment 1, 2), which were separated by tempered glass to prevent the permeation of nutrition, roots or microbes. The roots of selected cucumber seedlings (Section 2.2) were each artificially divided into two equal parts and planted in the two compartments, which contained 2 L sterile Hoagland nutrient solution. In the split-root hydroponic system, compartments were designed as follows: 1) CK (non-inoculation, compartment 1) þ (non-inoculation, compartment 2); 2) T (non-inoculation, compartment 1) þ (inoculated with Trichoderma harzianum T-E5 microconidia, compartment 2). There were 3 replicates of both the CK and T treatments, and each replicated system contained 6 cucumber seedlings. The entire experiment was carried out in a controlled-environment chamber at 30/26 C (day/night) and 60e70% humidity with a 16/8 h photoperiod at 4000 Lux light intensity. The cultivation systems were aerated daily to ensure the regular growth of cucumber seedlings. At the harvest day (20 days after inoculation), the shoots and roots of the plants in both CK and T were separated and then oven dried at 75 C to a constant weight, after which their dry weights were recorded. 2.4. Scanning electron microscopy of Trichoderma harzianum T-E5 colonization The colonization ability of Trichoderma harzianum T-E5 on cucumber roots was observed by SEM. The fresh root samples (1e2 cm) from CK and T (both collected from compartment 2) were processed before observation. Firstly, the samples were rinsed once with sterilized double-distilled water; then, the roots were fixed by immersion in 2.5% (v/v) glutaraldehyde for 8 h at 4 C, followed by washing 3e4 times with 0.1 M phosphate buffer for 15 min each. After that, the samples were dehydrated in a graded ethanol series (50%, 70%, 80%, 90% and 100%) and 100% ethanol (adding anhydrous Na2SO4) 15 min each step. Subsequently, samples were criticalpoint dried under liquid CO2 in a drier (Balzers SCD 020, Liechtenstein) and coated with goldepalladium in a sputter-coater (Balzers SCD 040, Liechtenstein). Finally, the prepared root samples were investigated and observed under a field emission SEM (HITACHI S-4800 FESEM, Tokyo, Japan).
2.2. Preparation of plant materials
2.5. Preparation of cucumber root exudates for organic acid analysis
Cucumber seeds (Cultivar Jinchun-No.4) were obtained from Tianjin Cucumber Research Center, China. The surface sterilization and pre-germination of the seeds were performed according to Hoyos-Carvajal et al. (2009). Subsequently, the germinated seeds were sown into a seedling-raising disk filled with a mixture of moist gnotobiotic quartz sand and vermiculite (1:1, v/v). The cucumber seedlings were watered with 1/4 Hoagland nutrient solution (Ca(NO3)2$4H2O, 945 mg/L; KNO3, 506 mg/L; NH4NO3, 80 mg/ L; KH2PO4, 136 mg/L; MgSO4$7H2O, 493 mg/L; Fe-EDTA, 20 mg/L; ZnSO4$7H2O, 1.7 mg/L; B4Na2O7$7H2O, 3.35 mg/L; CuSO4$5H2O, 0.25 mg/L and Na2MoO4, 0.12 mg/L) once a day until the first true
The plants have systemic induction to the stimulation of microorganisms (Vierheilig et al., 2003) through a plant-mediated mechanism; thus, the excreted cucumber root exudates in both compartments of the split-root hydroponic system were the same. However, the inoculated T-E5 in T may also secrete a certain amount of metabolites to affect the components of root exudates. Therefore, in order to effectively avoid the influence of inoculated microbial activity and decomposition on root exudates components, we selected to collect the cucumber root exudate solutions (2 L) in compartment 1 of both CK and T to ensure the accurate analysis of organic acids. The collected solutions were filtered
252
F. Zhang et al. / Plant Physiology and Biochemistry 83 (2014) 250e257
Fig. 1. (A) Illustration of the split-root hydroponic system; (B) The growth status of cucumber seedling at harvest day in CK and T. SEM (15.0 kV) images of cucumber root samples in CK (C, 200 ; D, 100 ) and T (E, 100 ; F, 100 ). Notes: In split-root hydroponic system: CK, (non-inoculation, compartment 1) þ (non-inoculation, compartment 2); T, (noninoculation, compartment 1) þ (inoculated with T. harzianum T-E5 microconidia, compartment 2).
through a 0.45 mm millipore filter and then eluted through a column, which contained XAD-4 resin (Sigma, USA) to absorb the lowmolecular-weight organic acids. Subsequently, the compounds absorbed on the resin were eluted by 200 ml spectral-grade methanol. The methanol solutions containing the compounds were evaporated in a rotary evaporator (Yarong model RE-52A, Shanghai, China) at 35 C for 15 min. Finally, all samples were concentrated in a volume of 5 ml sterilized double-distilled water. The concentrated solutions were stored at 20 C for subsequent analysis. 2.6. Dynamic quantification of T. harzianum in collected organic acid solutions One milliliter micro-conidial suspension of Trichoderma harzianum T-E5 (1.8 107 CFU mL1) was inoculated in two 50-fold
diluted organic acid solutions (CK and T, 100 ml) to a final concentration of approximately 1.8 105 CFU mL1. Then, the suspension was incubated at 28 C/170 rpm under aerated conditions. T. harzianum was quantified by dilution plating on Trichodermaselective medium (Elad et al., 1981) every 12 h after inoculation for 7 days. The biomass of T-E5 mycelium at 168th h was also recorded. 2.7. Identification and quantitative analysis of organic acids in root exudates Identification and quantification of organic acids were carried out by Agilent 1200 semi-preparative HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a UV detector. The separation of each sample was carried out on an XDB-C18 column (4.6 250 mm, Agilent, USA) at 35 C. After the initial column equilibration, analysis was performed by a gradient elution with
F. Zhang et al. / Plant Physiology and Biochemistry 83 (2014) 250e257
solvent A (5 mmol/L H2SO4, pH ¼ 4.6) and solvent B (methanol), with the following gradient profile: 0 min; 95% A plus 5% B (0.4 ml/ min)/15 min; 90% A plus 10% B (0.4 ml/min)/16 min; 90% A plus 10% B (0.5 ml/min)/20 min; 90% A plus 10% B (0.5 ml/min)/stop. The injection volume of each sample was 10 ml, and the UV detector wavelength was 210 nm. High purity oxalic acid, malic acid, citric acid, succinic acid and fumaric acid (Sigma, St Louis, Mo, USA) and chromatographically pure acetic acid (Kermel Co., Ltd, Tianjin, China) were used as the chemical standards for calibration. The mixed standard solution was prepared by dissolving 100 mg of each organic acid standard (oxalic acid, acetic acid, malic acid, citric acid, succinic acid and fumaric acid) in 100 ml ultrapure water. Then, the standard solution was diluted 100-fold and used as the mixed standard original liquor. The organic acids in the concentrated root exudates were identified and quantified by comparing their retention times and peak areas with standards. The quantitative results are expressed as mg (organic acids) g1 (root dry weight). Moreover, the presence of organic acids was confirmed using a liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) system (1200 series, Agilent, Santa Clara, CA, and ESI-MS, 6410 Triple Quad LC/ MS, Agilent, Santa Clara, CA). Both the positive-ion and negativeion modes were used to determine the molecular weights of the organic acids with mass spectra in the range of 50e500 m/z at a scan rate of 500 atomic mass units (amu)/s. 2.8. Mycelial growth and conidial germination of Trichoderma harzianum T-E5 under exogenous organic acid treatments An experiment was designed to study the influence of exogenous oxalic acid, malic acid and citric acid at different concentrations (0, 25, 50, 75 and 100 mM) on the mycelial growth and conidial germination of Trichoderma harzianum T-E5. Five-millimeter sameage T-E5 mycelium disks were inoculated in the center of 2% water agar petri dishes that contained organic acids at different concentrations. The T-E5 colony diameters were recorded 2 days after inoculation. To test the effects of different organic acids on the spore germination of T-E5, the T-E5 conidia suspension (1.8 107 CFU mL1) was firstly diluted to a magnitude of 103. Then, 100 ml of the diluted conidia suspension was spread onto 2% water agar petri dishes that contained different concentrations of organic acids. All petri dishes were incubated at 28 C in the dark. The number of germinating spores was recorded 3 days after incubation. Every treatment group consisted of three replicates, and in both experiments, water was used as a control instead of organic acids. 2.9. Colonization of Trichoderma harzianum T-E5 on cucumber roots under exogenous organic acid treatments To study the effects of different organic acids on root colonization by Trichoderma harzianum T-E5, the roots of cucumber plants with two true leaves were washed three times with sterilized distilled water and submerged in a series of concentrations (0, 25, 50, 75 and 100 mM) of each individual organic acid (oxalic acid, malic acid and citric acid) for 5 min. The roots treated with sterilized distilled water were used as a negative control. Then, each individual seedling was transplanted into different flasks (50 ml) filled with 40 ml of a 10-fold diluted conidial suspension of T. harzianum T-E5. The flasks with cucumber seedlings were placed on a slow shaker (75 rpm) in the greenhouse (Section 2.3) for 5 days. Then, the seedlings were harvested, and the roots were used for quantification of the T. harzianum T-E5 induced by exposure to different organic acids. The method for quantification of T. harzianum T-E5 was carried out according to Zhang et al. (2013b).
253
The roots were gently rinsed in sterile distilled water one time. One gram of fresh root sample from each treatment was triturated with mortar and pestle in sterilized distilled water. Then, the homogenate was diluted and plated on Trichoderma selective medium. The colony forming units (CFU) were counted after incubating the plates for 2 days at 28 C. Each treatment was conducted in three replications. 2.10. Statistical analysis Both the hydroponic and in vitro experiments were performed in triplicate. The means and standard deviations of all data were calculated using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) was used for statistical analyses, and significant differences were assessed using Duncan's multiple range tests at 95%-confidence level. Sigmaplot 11.0 was employed to illustrate all figures. 3. Results 3.1. Evaluation of cucumber plant growth in split-root hydroponic system The cucumber plants were all harvested and measured at the same time (20 days after inoculation, Fig. 1B), and significant differences (P 0.05) were detected between CK and T (Table 1). The average shoot height in T was increased by 63.7%, relative to CK. Similarly, the shoot and root dry weights of the cucumber plants were increased by 55.1 and 36.1%, respectively, compared with those in CK. 3.2. SEM study for colonization capacity of Trichoderma harzianum T-E5 The colonization observation of T-E5 on cucumber roots in the split-root hydroponic experiment was visualized by SEM. There were no Trichoderma hyphae present in CK, including the root tips and other parts of the cucumber roots (Figs. 1C, D). In contrast, obvious T. harzianum T-E5 hyphae were observed on the root surfaces of plants in T. To be specific, T. harzianum T-E5 mainly colonized the elongation and differentiation zones of cucumber root tips, while no T-E5 hyphae were observed at the tops of the root tips (Fig. 1E). In addition to root tips, many T-E5 hyphae also survived on other parts of the whole root surface (Fig. 1F). 3.3. Dynamic growth of Trichoderma harzianum T-E5 in organic acid solutions Trichoderma harzianum T-E5 had almost the same dynamic growth tendency in the collected dilute organic acids solutions
Table 1 Effects of different treatments on the biomass of cucumber plants for the whole split-root hydroponic system (compartment 1 þ 2 for both CK and T) on day of the harvest (20 d after inoculation). Treatments
Shoot height (cm)
Shoot dry weight (g/plant)
Root dry weight (g/plant)
CK T
10.2 ± 0.85b 16.7 ± 1.02a
1.78 ± 0.065b 2.76 ± 0.054a
0.083 ± 0.004b 0.113 ± 0.007a
Notes: In split-root hydroponic system: CK, (non-inoculation, compartment 1) þ (non-inoculation, compartment 2); T, (non-inoculation, compartment 1) þ (inoculated with T. harzianum T-E5 microconidia, compartment 2). Data were subjected to Duncan's analysis of variance (ANOVA). All values are the means of three replicates. Values with a different letter within the same column are significantly different at P 0.05 according to Duncan's multiple range tests. Numbers followed by “±”are the standard errors (SEs).
254
F. Zhang et al. / Plant Physiology and Biochemistry 83 (2014) 250e257
(Fig. 2A). Specifically, the number of T-E5 in CK and T were similar at the beginning of inoculation, with values of 9.6 and 11.6 (104 CFU mL1), respectively. Both increased rapidly after inoculation and successively reached the maximum level at the 72nd h (T, 182 104 CFU mL1) and the 84th h (CK, 163 104 CFU mL1). During the rapid growth, the T-E5 number in T was significantly higher than that in CK at all sampling times (36e84 h). Subsequently, the T-E5 number slowly decreased over several hours and remained at an approximately constant quantity at the 168th h. Meanwhile, T-E5 reached biomasses of 0.307 and 0.673 g L1 at the 168th h in CK and T, respectively (Fig. 2B). Though the biomass of TE5 was low in both CK and T, the T-E5 biomass in T was approximately twice that in CK, and this difference was significant. 3.4. Identification of organic acids in the root exudates by HPLC and LC/ESI-MS To determine and confirm the presence of organic acids in the concentrated root exudates, samples were subjected to HPLCcoupled LC/ESI-MS analysis. Different profiles and multiple peaks were detected in the chromatogram results of the two root exudates (Fig. 3). According to the retention times of the six organic acid standards (Fig. 3A), there were no qualitative differences between CK (Figs. 3B) and T (Fig. 3C). Three organic acids (oxalic acid, malic acid and citric acid) were identified from both CK and T. All
Fig. 3. The chromatograms of the organic acids in standard chemicals and root exudates: 1, oxalic acid; 2, malic acid; 3, acetic acid; 4, citric acid; 5, succinic acid; 6, fumaric acid. A) The chromatogram of standard organic acids. B, C) The chromatogram of the root exudates from the CK (B) and T (C) treatment groups. Notes: In split-root hydroponic system: CK, (non-inoculation, compartment 1) þ (non-inoculation, compartment 2); T, (non-inoculation, compartment 1) þ (inoculated with T. harzianum T-E5 microconidia, compartment 2).
corresponding peaks with retention times of 6.627, 8.378 and 14.820 were collected for confirmation by LC/ESI-MS. The data from LC/ESI-MS analysis are shown in Table 2. The mass spectra of peak 1 demonstrated a molecular mass of 89.1 Da [MH] in the negativeion mode; thus, peak 1 was identified as oxalic acid with a molecular weight of 90.04 Da. Similarly, the spectra of peak 2 showed a m/z ratio of 133.1 Da in the negative-ion mode and a molecular mass of 156.8 Da [M þ Na]þ under the positive-ion mode condition. The spectra of 4 exhibited a molecular mass of 214.8 Da [M þ Na]þ in the positive-ion mode. Therefore, peaks 2 and 4 were identified as malic acid and citric acid with molecular weights of 134.09 and 192.14 Da, respectively. The results overlapped with the HPLC analysis, and the collected root exudates had three organic acids in both the CK and T treatments. 3.5. Quantification of organic acids in the cucumber root exudates Fig. 2. (A) Dynamic growth of T. harzianum T-E5 in organic acid solutions 0e168 h after inoculation; (B) Biomass of T. harzianum T-E5 at 168th h. Notes: In split-root hydroponic system: CK, (non-inoculation, compartment 1) þ (non-inoculation, compartment 2); T, (non-inoculation, compartment 1) þ (inoculated with T. harzianum T-E5 microconidia, compartment 2).
The three organic acids identified were quantified by HPLC. As shown in Table 3, the amount of oxalic acid was the highest among the three organic acids. Moreover, the amount of released oxalic
F. Zhang et al. / Plant Physiology and Biochemistry 83 (2014) 250e257
255
Table 2 The liquid chromatography/electrospray ionization-mass spectrometry (LC/ESIMS) analysis of the peaks shown in Fig. 3B, C. Peak number
Retention time
Productions Negative
1 2 4
6.627 8.378 14.820
[M H] [M H]
m/z Positive [M þ Na]þ [M þ Na]þ
acid in T was approximately equivalent to 3 times that released in CK. The malic acid present in T was significantly increased by 73.03% compared with that in CK. However, there was no significant difference in citric acid amounts between CK and T. 3.6. Effects of organic acids on mycelial growth and conidial germination of Trichoderma harzianum T-E5 in vitro Exogenous applications of the previously detected organic acids (oxalic acid, malic acid and citric acid) exhibited different but significant stimulation effects on the mycelial growth of T-E5 (Fig. 4A). For oxalic acid, there was no significant difference between the stimulation rates (13.5e19.7%) at different concentrations (25e100 mM), and the colony diameter of T-E5 reached its greatest value of 7.10 cm under a 75 mM oxalic acid concentration. The T-E5 colony diameter reached its maximum value of 7.33 cm at 100 mM malic acid concentration. Moreover, the promotion percentage under 100 mM (23.6%) was significantly (P 0.05) higher than those at concentrations ranging from 25 to 75 mM (9.6e17.4%). Citric acid had greatest promotion effect (21.9%) and largest colony diameter (T-E5, 7.23 cm) at a concentration of 100 mM. Furthermore, it had the same promotion tendency with malic acid that the promotion percentage under 100 mM (21.9%) was significantly (P 0.05) higher than those at concentrations ranging from 25 to 75 mM (7.9e14.7%). In comparison with the control (0 mM), applied exogenous three organic acids significantly promoted conidial germination of T-E5 at any given concentration (25e100 mM) (Fig. 4B). Moreover, the conidial germination of T-E5 was more sensitive than its mycelial growth to the concentrations (25, 50, 75, 100 mM) of organic acids. In different oxalic acid treatments, the numbers of germinated T-E5 spores were significantly higher than the control, with stimulations ranging from 79.8% to 136.8% under concentrations ranging from 25 to 100 mM. The significant stimulation rates of malic and citric acid also increased gradually with increasing concentration until 100 mM, which generated the greatest promotion effects (161.4% in malic acid and 140.3% in citric acid) on T-E5 conidial germination.
Negative 89.1 133.1
Molecular weight
Compound
90.04 134.09 192.14
Oxalic acid Malic acid Citric acid
Positive 156.8 214.8
harzianum T-E5 colonization. The population density of T-E5 colonized on the cucumber root surface or the inner root tissues were recorded by dilution plate counting at 24, 48, 72, 96 and 120 h after inoculation. As shown in Table 4, all 3 organic acids significantly stimulated the colonization of T. harzianum T-E5 compared with the control (0 mM) at any sampling time. Specifically, oxalic acid exhibited a common promotion effect, and no significant difference was found among different concentrations ranging from 25 to 100 mM at any time point. For citric acid, the dynamic tendency of the T-E5 numbers was the same as those induced by oxalic acid (24e96 h). Subsequently, the colonization number of T-E5 at a concentration of 100 mM (11.77 104 cfu g1) was noticeably increased compared with those at 25e75 mM concentrations. Malic acid exhibited the most positive effect on the colonization of T-E5 among the 3 organic acids. When the concentration of malic acid was 100 mM, the population of T-E5 was significantly higher than those at concentrations of 25e75 mM at the 96th and 120th h. 4. Discussion In this study, we present the results of in vitro and in plant experiments regarding plant growth, fungal colonization and population dynamics, and chemical analysis. The results showed
3.7. Quantification of Trichoderma harzianum T-E5 in cucumber roots management with different organic acids A hydroponic experiment was performed to evaluate the positive effects of previously detected organic acids on Trichoderma Table 3 The quantitative determination of organic acids in cucumber root exudates by HPLC. Treatments
CK T
Organic acids (mg.g-1 root DW) Oxalic acid
Malic acid
Citric acid
1.81 ± 0.17b 4.78 ± 0.22a
0.89 ± 0.07b 1.54 ± 0.08a
0.96 ± 0.12a 1.11 ± 0.17a
Notes: In split-root hydroponic system: CK, (non-inoculation, compartment 1) þ (non-inoculation, compartment 2); T, (non-inoculation, compartment 1) þ (inoculated with T. harzianum T-E5 microconidia, compartment 2). All values are the means of three replicates. Values with different letters within the same column are significantly different at P 0.05 according to Duncan's test. Numbers followed by “±”are the standard errors (SEs).
Fig. 4. Effects of exogenous organic acid treatments on (A) mycelial growth and (B) conidial germination of T. harzianum T-E5. Notes: In split-root hydroponic system: CK, (non-inoculation, compartment 1) þ (non-inoculation, compartment 2); T, (noninoculation, compartment 1) þ (inoculated with T. harzianum T-E5 microconidia, compartment 2). Data were analyzed by Duncan's ANOVA test. Error bars represent the standard deviation of three replicates.
256
F. Zhang et al. / Plant Physiology and Biochemistry 83 (2014) 250e257
Table 4 T. harzianum T-E5 population in cucumber roots under different organic acid treatments 24e120 h after inoculation. Treatments
Concentration (mM)
104 T. harzianum cfu g1 root dry weight 24 h
Oxalic acid
Malic acid
Citric acid
0 25 50 75 100 0 25 50 75 100 0 25 50 75 100
1.36 2.68 2.79 2.91 2.89 1.36 2.24 2.38 2.42 2.49 1.36 2.37 2.46 2.45 2.59
48 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.28b 0.26a 0.22a 0.33a 0.31a 0.28b 0.32a 0.27a 0.29a 0.36a 0.28b 0.19a 0.34a 0.26a 0.29a
1.75 3.77 3.98 4.11 4.23 1.75 3.31 3.58 3.66 3.89 1.75 3.47 3.65 3.75 3.97
72 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.35b 0.36a 0.42a 0.53a 0.51a 0.35b 0.29a 0.31a 0.35a 0.42a 0.35b 0.32a 0.39a 0.33a 0.41a
2.69 4.89 5.47 6.46 6.72 2.69 4.56 4.97 5.92 6.86 2.69 4.31 4.78 5.46 6.44
96 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.29b 0.57a 0.62a 0.72a 0.69a 0.29b 0.52a 0.59a 0.63a 0.71a 0.29b 0.49a 0.55a 0.61a 0.68a
3.57 5.71 6.55 7.93 8.66 3.77 5.33 5.94 7.36 9.64 3.77 5.21 5.91 7.22 8.76
120 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.33b 0.64a 0.69a 0.82a 0.91a 0.33c 0.57b 0.68b 0.81b 0.94a 0.33b 0.59a 0.66a 0.79a 0.89a
4.29 6.76 7.87 9.46 10.56 4.59 6.27 6.81 8.56 13.06 4.59 6.03 6.89 8.75 11.77
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.51b 0.72a 0.89a 1.03a 0.98a 0.51c 0.67b 0.70b 0.93b 1.31a 0.51d 0.62c 0.72c 0.93b 1.04a
Notes: All values are the means of three replicates. Values with a different letter within the same column are significantly different at P 0.05 according to Duncan's test. Numbers followed by “±”are the standard errors (SEs).
that three organic acids (oxalic acid, malic acid and citric acid) facilitated the Trichoderma harzianum T-E5 colonization of cucumber roots. The inoculation of Trichoderma harzianum T-E5 significantly increased the biomass (shoot height and dry weights) of the cucumber plants compared with the control (CK) in the split-root hydroponic system (Table 1), which was consistent with the previous result of Yedidia et al. (2001). The report of Shivanna et al. (2005) showed that the promotion effect of Trichoderma depends on many elements, and the root colonization ability is recognized as one of the most important characteristics. Harman et al. (2004) also demonstrated that colonization by Trichoderma is beneficial to plants growth. Thus, we further observed the colonization status of T. harzianum T-E5 on the cucumber root surface by SEM (Figs. 1E, F). The hyphae of T-E5 densely covered the cucumber roots. Trichoderma spp. could efficiently colonize the root surface and, subsequently, on the elongation zone of the roots (Harman, 2000). Certain compounds in the root exudates could provide an important nutritional resource for the growth of Trichoderma to facilitate its root colonization (Vargas et al., 2009). A split-root hydroponic experiment based on plants systemic induction was designed for the collecting of cucumber root exudates in our research. Systemic induction of plant actions by microorganisms was also reported in the study of Scheffknecht et al. (2006). The cucumber root exudate solutions in compartment 1 of both CK and T were collected separately for organic acids analysis. Trichoderma harzianum T-E5 grew more rapidly and produced more mycelium in the organic acid solution collected from T than those found in CK (Fig. 2). This result indicates that T organic acid solution might provide more nutrient sources utilized by T. harzianum T-E5 to support its growth. Therefore, we further determined the compositions of the two organic acids solutions collected, and the presence of three organic acids (oxalic acid, malic acid and citric acid) were confirmed by HPLC and LC/ESI-MS analyses (Fig. 3, Table 2). Moreover, the identified organic acids were quantified in the CK and T solutions, and the results demonstrated that the secreted organic acid levels in T were significantly higher than those in CK. This result well explained the above result that the T-E5 population in T was significantly higher than in CK (Fig. 2). Many reports provide evidence to highlight the role of organic acids, noting that the overproduction of citric and malic acids can cause an increased colonization of mycorrhizal fungi and rhizobacteria in tobacco and alfalfa plants (Tesfaye et al., 2003). To our knowledge, few previous reports have demonstrated that organic acids in cucumber root exudates can function as carbon sources to
attract the colonization of Trichoderma on cucumber roots. The results obtained in vitro indicated that the three organic acids had different degrees of promotional effects on the mycelial growth and conidial germination of T-E5. Our result was consistent with those of Leonian and Lilly (1940) and showed that organic acids could effectively promote the growth of fungi. Nelson et al. (1988) also suggested that the biocontrol effect of Trichoderma koningii on Pythium seed rot mainly resulted from the addition of organic acids that stimulated the growth of Trichoderma strains. However, the reported stimulation effects were not absolutely positive for all concentrations of organic acids. In the present study, the mycelial growth and conidial germination of T. harzianum T-E5 reached their maxima when the concentrations of malic and citric acids were both 100 mM, which essentially showed that the high concentrations of organic acids provided more carbon sources for T-E5 utilization. For oxalic acid, T. harzianum T-E5 was more efficient at a concentration of 75 mM, although 100 mM offered a greater carbon source. Morton and Macmillan (1954) explained that the promotion differences mainly occurred because organic acids reduced the pH of the culture medium. T. harzianum T-E5 was most likely more sensitive to the acidity of oxalic acid than the other two organic acids (malic acid and citric acid). In other words, both the carbon source and medium pH had important influences on the growth of strain T-E5 under oxalic acid treatment. The influences of different organic acids on the dynamic colonization numbers of Trichoderma harzianum T-E5 in cucumber roots were also determined in a hydroponic system. The colonization populations of T-E5 gradually increased, along with the increased organic acid concentrations and extended culture times (Table 4). In this relatively open hydroponic environment, the influence of the surrounding acidity was less significant than nutrient supplement on the colonization number of T-E5 in cucumber roots. Moreover, the chosen culture time was appropriate because no significant decline was observed in the T-E5 population during the culture process, ensuring an accurate investigation of colonization in cucumber roots. 5. Conclusion Beneficial Trichoderma harzianum T-E5 had potential promoting effects for cucumber plant growth in split-root hydroponic system, mainly due to their efficient colonization on the cucumber root surface. The amounts of organic acids in T were significantly higher than in CK. The present study revealed that there was a strong correlation between organic acid utilization and root colonization.
F. Zhang et al. / Plant Physiology and Biochemistry 83 (2014) 250e257
After a series of in vitro and hydroponic system experiments, organic acids were proven to be used as nutritional sources by Trichoderma harzianum T-E5 to contribute to fungal population colonization on cucumber roots. Acknowledgements This work was financially supported by Innovative Research Team Development Plan of the Ministry of Education of China (IRT1256), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, the 111 project (B12009), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the Chinese Ministry of Science and Technology (2011BAD11B03), the Chinese Ministry of Agriculture (201103004). Contributions The contribution of the paper: Illustration the role of organic acids in root exudates could be performed as carbon sources to attract the colonization of Trichoderma on cucumber roots. Author Contributions: Conceived and designed the experiments: WR, FZ, QS. Performed the experiments: FZ, XM, XY. Analyzed the data: FZ. Contributed reagents/materials/analysis tools: WR, QS. Wrote the paper: FZ, WR. This research is an important and original work. Trichoderma species are well-known by their abilities of promoting plants growth and effective biocontrol of various soil-borne diseases. However, the survival rate and population density of Trichoderma colonized in the rhizosphere are the prerequisites for their effectiveness or specific functions in practice. To our knowledge, little research works had been developed to unravel the role of organic acids in root exudates could be performed as carbon sources to attract the colonization of Trichoderma on cucumber roots. We confirmed the organic acids could be used as nutritional basis of Trichoderma spp. to stimulate its colonization populations on cucumber roots in our manuscript carefully. Thus, the manuscript is an original contribution to this area. The paper may be of particular interest to the readers of your journal. References €m, S., 2000. Root-colonizing fungi from oilseed rape and their inhibition of Alstro Verticillium dahliae. J. Phytopathol. 148, 417e423. Badri, D., Vivanco, J.M., 2009. Regulation and function of root exudates. Plant. Cell. Environ. 32, 666e681. Bae, H., Sicher, R.C., Kim, M.S., Kim, S.H., Strem, M.D., Melnick, R.L., Bailey, B.A., 2009. The beneficial endophyte Trichoderma hamatum isolate DIS 219b promotes growth and delays the onset of the drought response in Theobroma cacao. J. Exp. Bot. 60, 3279e3295. Bailey, B.A., Bae, H., Strem, M.D., Roberts, D.P., Thomas, S.E., Crozier, J., Samuels, G.J., Choi, I.Y., Holmes, K.A., 2006. Fungal and plant gene expression during the colonization of cacao seedlings by endophytic isolates of four Trichoderma species. Planta 224, 1449e1464. Brotman, Y., Landau, U., Cuadros-Inostroza, A., Takayuki, T., Fernie, A.R., Chet, I., Viterbo, A., Willmitzer, L., 2013. Trichoderma-plant root colonization: escaping early plant defense responses and activation of the antioxidant machinery for saline stress tolerance. Plos. Pathog. 9. Chen, L.H., Huang, X.Q., Zhang, F.G., Zhao, D.K., Yang, X.M., Shen, Q.R., 2012. Application of Trichoderma harzianum SQR-T037 bio-organic fertiliser significantly controls Fusarium wilt and affects the microbial communities of continuously cropped soil of cucumber. J. Sci. Food. Agr. 92. s-Penagos, C., Lo pez-Bucio, J., Contreras-Cornejo, H.A., Macías-Rodríguez, L.I., Corte 2009. Trichoderma virens, a plant beneficial fungus, enhances biomass production and promotes lateral root growth through an auxin-dependent mechanism in Arabidopsis. Plant. Physiol. 149, 1579e1592. Elad, Y., Chet, I., Henis, Y., 1981. A selective medium for improving quantitative isolation of Trichoderma spp. from soil. Phyloparasitica 9, 59e67. Hao, W.Y., Ren, L.X., Ran, W., Shen, Q.R., 2010. Allelopathic effects of root exudates from watermelon and rice plants on Fusarium oxysporum f.sp. niveum. Plant. Soil. 336, 485e497. Harman, G.E., 2000. Combining effective strains of Trichoderma harzianum and solid matrix priming to improve biological seed treatments. Plant. Dis. 84, 377e393.
257
Harman, G.E., Howell, C.R., Viterbo, A., Chet, I., Lorito, M., 2004. Trichoderma speciesoportunistic, avirulent plant symbionts. Nat. Rev. Microbiol. 2, 43e56. Hiltner, L., 1904. Ueber neuere Erfahrungen und Probleme auf dem Gebiete der Bodenbakteriologie und unter besonderer BerUcksichtigung der Grundungung und Brache. Arb. Deut. Landw. Gesell 98, 59e78. Howell, C.R., 2002. Cotton seedling preemergence damping-off incited by Rhizopus oryzae and Pythium spp and its biological control with Trichoderma spp. Phytopathology 92, 177e180. Hoyos-Carvajal, L., Orduz, S., Bissett, J., 2009. Growth stimulation in bean (Phaseolus vulgaris L.) by Trichoderma. Biocontrol 51, 409e416. Huang, X.Q., Chen, L.H., Ran, W., Shen, Q.R., Yang, X.M., 2011. Trichoderma harzianum strain SQR-T37 and its bio-organic fertilizer could control Rhizoctonia solani damping-off disease in cucumber seedlings mainly by the mycoparasitism. Appl. Microbiol. Biot. 91, 741e755. Leonian, L.H., Lilly, V.G., 1940. Studies on the nutrition of fungi. IV. Factors influencing the growth of some thiamine-requiring fungi. Am. J. Bot. 27, 18e26. Lilia, C.C., Paul, G.D., Dmitri, F., Mohammad-Reza, H., Rainer, B., Nicolaus, V.W., 2011. Root exudation of sugars, amino acids, and organic acids by maize as affected by nitrogen, phosphorus, potassium, and iron deficiency. J. Plant. Nutr. Soil. Sci. 174, 3e11. Ling, N., Raza, W., Ma, J.H., Huang, Q.W., Shen, Q.R., 2011. Identification and role of organic acids in watermelon root exudates for recruiting Paenibacillus polymyxa SQR-21 in the rhizosphere. Eur. J. Soil. Biol. 47, 374e379. Ling, N., Huang, Q.W., Guo, S.W., Shen, Q.R., 2011. Paenibacillus polymyxa SQR-21 systemically affects root exudates of watermelon to decrease the conidial germination of Fusarium oxysporum f. sp. niveum. Plant. Soil. 341, 485e493. Liu, F., Wen, X.S., 2006. Progress in relationship between root exudates and rhizospheric microorganism. Food. Drug. 8, 37e40. pez-Bucio, J., Nieto-Jacobo, M.F., Ramírez-Rodríguez, V., Herrera-Estrella, L., 2000. Lo Organic acid metabolism in plants: from adaptive physiology to transgenic varieties for cultivation in extreme soils. Plant. Sci. 160, 1e13. Mogan, J.A.W., Bending, G.D., White, P.J., 2005. Biological costs and benefits to plant-microbe interactions in the rhizosphere. J. Exp. Bot. 56, 729e1739. Morton, A.G., Macmillan, A., 1954. The assimilation of nitrogen from ammonium salts and nitrate by fungi. J. Exp. Bot. 5, 232e252. Nelson, E.B., Harman, G.E., Nash, G.T., 1988. Enhancement of Trichoderma-induced biological control of pythium seed rot and pre-emergence dramping-off of peas. Soil. Biol. Biochem 20, 145e150. Raaijmakers, J.M., Paulitz, T.C., Steinberg, C., Alabouvette, C., Moenne-Loccoz, Y., 2009. The rhizosphere: a playground and battlefield for soilborne pathogens and beneficial microorganisms. Plant. Soil. 321, 341e361. Rabeendran, N., Jones, E.E., Moot, D.J., Stewart, A., 2006. Biocontrol of Sclerotinia lettuce drop by Coniothyrium minitans and Trichoderma hamatum. Biol. Control 39, 352e362. , L.B., 2009. The role of microbial Randy, O.C., Hexon, A.C.C., Lourdes, M.R., Jose signals in plant growth and development. Plant. Signal. Behav. 4, 701e712. Rigot, J., Matsumura, F., 2002. Assessment of the rhizosphere competency and pentachlorophenol- metabolizing activity of a pesticide-degrading strain of Trichoderma harzianum introduced into the root zone of corn seedlings. J. Environ. Sci. Heal. B 37, 201e210. Scheffknecht, S., Mammerler, R., Steinkellner, S., Vierheilig, H., 2006. Root exudates of mycorrhizal tomato plants exhibit a different effect on microconidia germination of Fusarium oxysporum f. sp. lycopersici than root exudates from nonmycorrhizal tomato plants. Mycorrhiza 16, 365e370. Shivanna, M.B., Meera, M.S., Kubota, M., Hyakumachi, M., 2005. Promotion of growth and yield in cucumber by zoysiagrass rhizosphere fungi. Microbes. Environ. 2, 34e40. Swarnalee, D., Rani, T.S., Appa, R.P., 2013. Root exudate-Induced alterations in Bacillus cereus cell wall contribute to root colonization and plant growth promotion. Plos. One. http://dx.doi.org/10.1371/journal.pone.0078369. Tan, S.Y., Yang, C.L., Mei, X.L., Shen, S., Raza, W., Shen, Q.R., Xu, Y.C., 2013. The effect of organic acids from tomato root exudates on rhizosphere colonization of Bacillus amyloliquefaciens T-5. Appl. Soil. Ecol. 64, 15e22. Tesfaye, M., Dufault, N.S., Dornbusch, M., Allan, D., Vance, C.P., Samac, D.A., 2003. Influence of enhanced malate dehydrogenase expression by alfalfa on diversity of rhizobacteria and soil nutrient availability. Soil. Biol. Biochem 35, 1103e1113. Vargas, W.A., Mandawe, J.C., Kenerley, C.M., 2009. Plant-derived sucrose is a key element in the symbiotic association between Trichoderma virens and maize plants. Plant. Physiol. 151, 792e808. , Y., 2003. Systemic inhibition of arbuscular mycorrhiza Vierheilig, H., Lerat, S., Piche development by root exudates of cucumber plants colonized by Glomus mosseae. Mycorrhiza 13, 167e170. Yedidia, I., Srivastva, A.K., Kapulnik, Y., Chet, I., 2001. Effect of Trichoderma harzianum on microelement concentrations and increased growth of cucumber plants. Plant. Soil. 235, 235e242. Zhang, F.G., Zhu, Z., Yang, X.M., Ran, W., Shen, Q.R., 2013. Trichoderma harzianum TE5 significantly affects cucumber root exudates and fungal community in the cucumber rhizosphere. Appl. Soil. Ecol. 72, 41e48. Zhang, F.G., Yuan, J., Yang, X.M., Cui, Y.Q., Chen, L.H., Ran, W., Shen, Q.R., 2013. Putative Trichoderma harzianum mutant promotes cucumber growth by enhanced production of indole acetic acid and plant colonization. Plant. Soil. 368, 433e444. Zhang, F.G., Zhu, Z., Wang, B.B., Wang, P., Yu, G.H., Wu, M.J., Chen, W., Ran, W., Shen, Q.R., 2013. Optimization of Trichoderma harzianum T-E5 biomass and determining the degradation sequence of biopolymers by FTIR in solid-state fermentation. Ind. Crop. Prod. 49, 619e627.
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Quantification and role of organic acids in cucumber root exudates in Trichoderma harzianum T-E5 colonization Fengge Zhang a, Xiaohui Meng a, Xingming Yang a, b, Wei Ran a, b, *, Qirong Shen a, b a b
National Engineering Research Center for Organic-based Fertilizers, Nanjing Agricultural University, Nanjing 210095, PR China Jiangsu Collaborative Innovation Center for Solid Organic Waste Utilization, Nanjing Agricultural University, Nanjing 210095, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 April 2014 Accepted 14 August 2014 Available online 23 August 2014
The ability to colonize on plant roots is recognized as one of the most important characteristics of the beneficial fungi Trichoderma spp. The aim of this study is to prove that the utilization of organic acids is a major trait of Trichoderma harzianum T-E5 for colonization of cucumber roots. A series experiments in split-root hydroponic system and in vitro were designed to demonstrate the association between the utilization of organic acids and T-E5 colonization on cucumber roots. In the split-root hydroponic system, inoculation with T-E5 (T) significantly increased the biomass of cucumber plants compared with CK (non-inoculation with T-E5). The T-E5 hyphae densely covering the cucumber root surface were observed by scanning electron microscopy (SEM). Three organic acids (oxalic acid, malic acid and citric acid) were identified from both the CK and T treatments by HPLC and LC/ESI-MS procedures. The amounts of oxalic acid and malic acid in T were significantly higher than those in CK. All the organic acids exhibited different and significant stimulation effects on the mycelial growth and conidial germination of T-E5 in vitro. An additional hydroponic experiment demonstrated the positive effects of organic acids on the T-E5 colonization of cucumber roots. In conclusion, the present study revealed that certain organic acids could be used as nutritional sources for Trichoderma harzianum T-E5 to reinforce its population on cucumber roots. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Colonization HPLC Nutritional source Organic acids Split-root hydroponic system
1. Introduction Trichoderma spp., as well-known rhizosphere-resident antagonistic fungus, is widely used to control plant diseases caused by numerous soil-borne pathogens, such as Rhizoctonia solani (Huang et al., 2011), Rhizopus oryzae (Howell, 2002), Fusarium spp. (Zhang et al., 2013a), etc. Moreover, Trichoderma strains have the ability to promote plant growth and development by increasing plant nutrient uptake, photosynthetic and respiratory efficiency, and production of growth hormones (Contreras-Cornejo et al., 2009; Zhang et al., 2013b). Many reports also show that the combination of organic fertilizers and Trichoderma strains is a good way to control plant diseases or facilitate high-yield crops (Chen et al.,
Abbreviations: CFU, colony forming units; HPLC, high performance liquid chromatography; LC/ESI-MS, liquid chromatography/ electrospray ionization-mass spectrometry; PDA, potato dextrose agar; SEM, scanning electron microscopy; UV, ultra violet. * Corresponding author. Postal address: College of Resources and Environmental Sciences, Nanjing Agricultural University, 210095 Nanjing, Jiangsu Province, PR China. Tel.: þ86 025 84396212; fax: þ86 025 84396824. E-mail address: [email protected] (W. Ran). http://dx.doi.org/10.1016/j.plaphy.2014.08.011 0981-9428/© 2014 Elsevier Masson SAS. All rights reserved.
2012; Zhang et al., 2013c). Evidence has accumulated indicating that some Trichoderma strains have good colonization abilities (Rigot and Matsumura, 2002; Brotman et al., 2013) and can successfully survive on root surfaces and even on entire roots (Bailey et al., 2006; Bae et al., 2009). In practice, the survival rate and population density of beneficial microorganisms are the prerequisites for their effectiveness or specific functions in the rhizosphere (Raaijmakers et al., 2009). Moreover, the quality and effectiveness of biological agents are also determined by the continuous extension abilities of functional strains during the €m colonization of soil or plant roots in the field. For instance, Alstro € m, 2000) demonstrated that the biocontrol effect of Tricho(Alstro derma was directly dependent on its rhizosphere colonization ability, and Trichoderma proved able to colonize lettuce roots to successfully control the disease caused by Sclerotina minor in field experiments (Rabeendran et al., 2006). The rhizosphere is a dynamic system in which a number of interactions and interdependent relationships between plant roots and microorganisms take place (Mogan et al., 2005). Moreover, as the medium of roots and surrounding microorganisms, root exudates play an important role in information transmission between them. Different plants have different root exudates, and the root
F. Zhang et al. / Plant Physiology and Biochemistry 83 (2014) 250e257
exudates released to the rhizosphere during the process of plant growth include a wide range of organic and inorganic materials (Badri and Vivanco, 2009). The composition and quantity of these compounds directly affect the dominant species and breeding of microorganisms in the rhizosphere (Zhang et al., 2013a). The root exudates, such as sugars, amino acids, phenolic acids and organic acids (Swarnalee et al., 2013), have significant stimulatory or inhibitory effects on the growth of microorganisms (Hao et al., 2010; Ling et al., 2011a) and can be used as nutrients or signals by some beneficial microorganisms (Randy et al., 2009). The rhizosphere microorganisms are able to use soluble root exudates compounds as their major carbon nutrient source, which is considered to be the nutritional basis to guarantee successful rhizosphere colonization (Hiltner, 1904). Raaijmakers et al. (2009) also demonstrated that these root exudates are the main food sources for microbes and a driving force of their population density and activities. Low-molecular-weight organic acids are usually present in root exudates, of which they are reported as a main component (Liu and Wen, 2006). Many studies have focused on demonstrating the functions of organic acids involved in the metabolic processes of pez-Bucio et al., 2000; Lilia et al., 2011) and shown the plants (Lo chemotaxis response of bacteria toward different organic acids (Tan et al., 2013). However, few studies have investigated the effects of organic acids on the activities of rhizosphere fungi (for example, Trichoderma spp.) concerning the relationship between organic acids and colonization ability of fungi in the rhizosphere. In this study, we hypothesized that organic acids could be used as a nutritional basis for rhizosphere fungi (Trichoderma spp.) to support their colonization of cucumber roots. Therefore, our study aimed to investigate the colonization of Trichoderma harzianum TE5 hyphae on cucumber roots using scanning electron microscope (SEM) in a split-root hydroponic system; to identify and measure the organic acids in cucumber root exudates; and to confirm the role of organic acids (used as nutritional sources to attract T. harzianum) in T-E5 colonization of cucumber roots. 2. Methods 2.1. Microorganisms and microconidia Trichoderma harzianum strain T-E5 (CCTCC No.AF2012011, China Center for Type Culture Collection) was provided by Jiangsu Provincial Key Lab of Organic Solid Waste Utilization, Nanjing, China and used throughout this study. The strain was routinely incubated on potato dextrose agar (PDA) medium in Petri dishes in the dark at 28 C for 7 days and maintained at 80 C in 30% glycerol for longterm storage. The conidial suspension of Trichoderma harzianum TE5 was prepared according to Zhang et al. (2013a). The conidia concentration was 1.8 107 CFU mL1, which was determined by direct observation on a hemacytometer (Hinotek Technology, Zhongguancun, China).
251
leaf unfolded, which roughly took 7 days. Seedlings at a similar stage were carefully removed from the disk, and the roots were lightly washed 3 times with sterilized double-distilled water before they were moved into the split-root hydroponic system. 2.3. Description of the split-root hydroponic experiment A split-root hydroponic experiment was designed mainly for the followed cucumber root exudates experiment in this study. The split-root hydroponic system was employed according to Ling et al. (2011b), with a few modifications. The system consisted of two compartments (Fig. 1A, compartment 1, 2), which were separated by tempered glass to prevent the permeation of nutrition, roots or microbes. The roots of selected cucumber seedlings (Section 2.2) were each artificially divided into two equal parts and planted in the two compartments, which contained 2 L sterile Hoagland nutrient solution. In the split-root hydroponic system, compartments were designed as follows: 1) CK (non-inoculation, compartment 1) þ (non-inoculation, compartment 2); 2) T (non-inoculation, compartment 1) þ (inoculated with Trichoderma harzianum T-E5 microconidia, compartment 2). There were 3 replicates of both the CK and T treatments, and each replicated system contained 6 cucumber seedlings. The entire experiment was carried out in a controlled-environment chamber at 30/26 C (day/night) and 60e70% humidity with a 16/8 h photoperiod at 4000 Lux light intensity. The cultivation systems were aerated daily to ensure the regular growth of cucumber seedlings. At the harvest day (20 days after inoculation), the shoots and roots of the plants in both CK and T were separated and then oven dried at 75 C to a constant weight, after which their dry weights were recorded. 2.4. Scanning electron microscopy of Trichoderma harzianum T-E5 colonization The colonization ability of Trichoderma harzianum T-E5 on cucumber roots was observed by SEM. The fresh root samples (1e2 cm) from CK and T (both collected from compartment 2) were processed before observation. Firstly, the samples were rinsed once with sterilized double-distilled water; then, the roots were fixed by immersion in 2.5% (v/v) glutaraldehyde for 8 h at 4 C, followed by washing 3e4 times with 0.1 M phosphate buffer for 15 min each. After that, the samples were dehydrated in a graded ethanol series (50%, 70%, 80%, 90% and 100%) and 100% ethanol (adding anhydrous Na2SO4) 15 min each step. Subsequently, samples were criticalpoint dried under liquid CO2 in a drier (Balzers SCD 020, Liechtenstein) and coated with goldepalladium in a sputter-coater (Balzers SCD 040, Liechtenstein). Finally, the prepared root samples were investigated and observed under a field emission SEM (HITACHI S-4800 FESEM, Tokyo, Japan).
2.2. Preparation of plant materials
2.5. Preparation of cucumber root exudates for organic acid analysis
Cucumber seeds (Cultivar Jinchun-No.4) were obtained from Tianjin Cucumber Research Center, China. The surface sterilization and pre-germination of the seeds were performed according to Hoyos-Carvajal et al. (2009). Subsequently, the germinated seeds were sown into a seedling-raising disk filled with a mixture of moist gnotobiotic quartz sand and vermiculite (1:1, v/v). The cucumber seedlings were watered with 1/4 Hoagland nutrient solution (Ca(NO3)2$4H2O, 945 mg/L; KNO3, 506 mg/L; NH4NO3, 80 mg/ L; KH2PO4, 136 mg/L; MgSO4$7H2O, 493 mg/L; Fe-EDTA, 20 mg/L; ZnSO4$7H2O, 1.7 mg/L; B4Na2O7$7H2O, 3.35 mg/L; CuSO4$5H2O, 0.25 mg/L and Na2MoO4, 0.12 mg/L) once a day until the first true
The plants have systemic induction to the stimulation of microorganisms (Vierheilig et al., 2003) through a plant-mediated mechanism; thus, the excreted cucumber root exudates in both compartments of the split-root hydroponic system were the same. However, the inoculated T-E5 in T may also secrete a certain amount of metabolites to affect the components of root exudates. Therefore, in order to effectively avoid the influence of inoculated microbial activity and decomposition on root exudates components, we selected to collect the cucumber root exudate solutions (2 L) in compartment 1 of both CK and T to ensure the accurate analysis of organic acids. The collected solutions were filtered
252
F. Zhang et al. / Plant Physiology and Biochemistry 83 (2014) 250e257
Fig. 1. (A) Illustration of the split-root hydroponic system; (B) The growth status of cucumber seedling at harvest day in CK and T. SEM (15.0 kV) images of cucumber root samples in CK (C, 200 ; D, 100 ) and T (E, 100 ; F, 100 ). Notes: In split-root hydroponic system: CK, (non-inoculation, compartment 1) þ (non-inoculation, compartment 2); T, (noninoculation, compartment 1) þ (inoculated with T. harzianum T-E5 microconidia, compartment 2).
through a 0.45 mm millipore filter and then eluted through a column, which contained XAD-4 resin (Sigma, USA) to absorb the lowmolecular-weight organic acids. Subsequently, the compounds absorbed on the resin were eluted by 200 ml spectral-grade methanol. The methanol solutions containing the compounds were evaporated in a rotary evaporator (Yarong model RE-52A, Shanghai, China) at 35 C for 15 min. Finally, all samples were concentrated in a volume of 5 ml sterilized double-distilled water. The concentrated solutions were stored at 20 C for subsequent analysis. 2.6. Dynamic quantification of T. harzianum in collected organic acid solutions One milliliter micro-conidial suspension of Trichoderma harzianum T-E5 (1.8 107 CFU mL1) was inoculated in two 50-fold
diluted organic acid solutions (CK and T, 100 ml) to a final concentration of approximately 1.8 105 CFU mL1. Then, the suspension was incubated at 28 C/170 rpm under aerated conditions. T. harzianum was quantified by dilution plating on Trichodermaselective medium (Elad et al., 1981) every 12 h after inoculation for 7 days. The biomass of T-E5 mycelium at 168th h was also recorded. 2.7. Identification and quantitative analysis of organic acids in root exudates Identification and quantification of organic acids were carried out by Agilent 1200 semi-preparative HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a UV detector. The separation of each sample was carried out on an XDB-C18 column (4.6 250 mm, Agilent, USA) at 35 C. After the initial column equilibration, analysis was performed by a gradient elution with
F. Zhang et al. / Plant Physiology and Biochemistry 83 (2014) 250e257
solvent A (5 mmol/L H2SO4, pH ¼ 4.6) and solvent B (methanol), with the following gradient profile: 0 min; 95% A plus 5% B (0.4 ml/ min)/15 min; 90% A plus 10% B (0.4 ml/min)/16 min; 90% A plus 10% B (0.5 ml/min)/20 min; 90% A plus 10% B (0.5 ml/min)/stop. The injection volume of each sample was 10 ml, and the UV detector wavelength was 210 nm. High purity oxalic acid, malic acid, citric acid, succinic acid and fumaric acid (Sigma, St Louis, Mo, USA) and chromatographically pure acetic acid (Kermel Co., Ltd, Tianjin, China) were used as the chemical standards for calibration. The mixed standard solution was prepared by dissolving 100 mg of each organic acid standard (oxalic acid, acetic acid, malic acid, citric acid, succinic acid and fumaric acid) in 100 ml ultrapure water. Then, the standard solution was diluted 100-fold and used as the mixed standard original liquor. The organic acids in the concentrated root exudates were identified and quantified by comparing their retention times and peak areas with standards. The quantitative results are expressed as mg (organic acids) g1 (root dry weight). Moreover, the presence of organic acids was confirmed using a liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) system (1200 series, Agilent, Santa Clara, CA, and ESI-MS, 6410 Triple Quad LC/ MS, Agilent, Santa Clara, CA). Both the positive-ion and negativeion modes were used to determine the molecular weights of the organic acids with mass spectra in the range of 50e500 m/z at a scan rate of 500 atomic mass units (amu)/s. 2.8. Mycelial growth and conidial germination of Trichoderma harzianum T-E5 under exogenous organic acid treatments An experiment was designed to study the influence of exogenous oxalic acid, malic acid and citric acid at different concentrations (0, 25, 50, 75 and 100 mM) on the mycelial growth and conidial germination of Trichoderma harzianum T-E5. Five-millimeter sameage T-E5 mycelium disks were inoculated in the center of 2% water agar petri dishes that contained organic acids at different concentrations. The T-E5 colony diameters were recorded 2 days after inoculation. To test the effects of different organic acids on the spore germination of T-E5, the T-E5 conidia suspension (1.8 107 CFU mL1) was firstly diluted to a magnitude of 103. Then, 100 ml of the diluted conidia suspension was spread onto 2% water agar petri dishes that contained different concentrations of organic acids. All petri dishes were incubated at 28 C in the dark. The number of germinating spores was recorded 3 days after incubation. Every treatment group consisted of three replicates, and in both experiments, water was used as a control instead of organic acids. 2.9. Colonization of Trichoderma harzianum T-E5 on cucumber roots under exogenous organic acid treatments To study the effects of different organic acids on root colonization by Trichoderma harzianum T-E5, the roots of cucumber plants with two true leaves were washed three times with sterilized distilled water and submerged in a series of concentrations (0, 25, 50, 75 and 100 mM) of each individual organic acid (oxalic acid, malic acid and citric acid) for 5 min. The roots treated with sterilized distilled water were used as a negative control. Then, each individual seedling was transplanted into different flasks (50 ml) filled with 40 ml of a 10-fold diluted conidial suspension of T. harzianum T-E5. The flasks with cucumber seedlings were placed on a slow shaker (75 rpm) in the greenhouse (Section 2.3) for 5 days. Then, the seedlings were harvested, and the roots were used for quantification of the T. harzianum T-E5 induced by exposure to different organic acids. The method for quantification of T. harzianum T-E5 was carried out according to Zhang et al. (2013b).
253
The roots were gently rinsed in sterile distilled water one time. One gram of fresh root sample from each treatment was triturated with mortar and pestle in sterilized distilled water. Then, the homogenate was diluted and plated on Trichoderma selective medium. The colony forming units (CFU) were counted after incubating the plates for 2 days at 28 C. Each treatment was conducted in three replications. 2.10. Statistical analysis Both the hydroponic and in vitro experiments were performed in triplicate. The means and standard deviations of all data were calculated using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) was used for statistical analyses, and significant differences were assessed using Duncan's multiple range tests at 95%-confidence level. Sigmaplot 11.0 was employed to illustrate all figures. 3. Results 3.1. Evaluation of cucumber plant growth in split-root hydroponic system The cucumber plants were all harvested and measured at the same time (20 days after inoculation, Fig. 1B), and significant differences (P 0.05) were detected between CK and T (Table 1). The average shoot height in T was increased by 63.7%, relative to CK. Similarly, the shoot and root dry weights of the cucumber plants were increased by 55.1 and 36.1%, respectively, compared with those in CK. 3.2. SEM study for colonization capacity of Trichoderma harzianum T-E5 The colonization observation of T-E5 on cucumber roots in the split-root hydroponic experiment was visualized by SEM. There were no Trichoderma hyphae present in CK, including the root tips and other parts of the cucumber roots (Figs. 1C, D). In contrast, obvious T. harzianum T-E5 hyphae were observed on the root surfaces of plants in T. To be specific, T. harzianum T-E5 mainly colonized the elongation and differentiation zones of cucumber root tips, while no T-E5 hyphae were observed at the tops of the root tips (Fig. 1E). In addition to root tips, many T-E5 hyphae also survived on other parts of the whole root surface (Fig. 1F). 3.3. Dynamic growth of Trichoderma harzianum T-E5 in organic acid solutions Trichoderma harzianum T-E5 had almost the same dynamic growth tendency in the collected dilute organic acids solutions
Table 1 Effects of different treatments on the biomass of cucumber plants for the whole split-root hydroponic system (compartment 1 þ 2 for both CK and T) on day of the harvest (20 d after inoculation). Treatments
Shoot height (cm)
Shoot dry weight (g/plant)
Root dry weight (g/plant)
CK T
10.2 ± 0.85b 16.7 ± 1.02a
1.78 ± 0.065b 2.76 ± 0.054a
0.083 ± 0.004b 0.113 ± 0.007a
Notes: In split-root hydroponic system: CK, (non-inoculation, compartment 1) þ (non-inoculation, compartment 2); T, (non-inoculation, compartment 1) þ (inoculated with T. harzianum T-E5 microconidia, compartment 2). Data were subjected to Duncan's analysis of variance (ANOVA). All values are the means of three replicates. Values with a different letter within the same column are significantly different at P 0.05 according to Duncan's multiple range tests. Numbers followed by “±”are the standard errors (SEs).
254
F. Zhang et al. / Plant Physiology and Biochemistry 83 (2014) 250e257
(Fig. 2A). Specifically, the number of T-E5 in CK and T were similar at the beginning of inoculation, with values of 9.6 and 11.6 (104 CFU mL1), respectively. Both increased rapidly after inoculation and successively reached the maximum level at the 72nd h (T, 182 104 CFU mL1) and the 84th h (CK, 163 104 CFU mL1). During the rapid growth, the T-E5 number in T was significantly higher than that in CK at all sampling times (36e84 h). Subsequently, the T-E5 number slowly decreased over several hours and remained at an approximately constant quantity at the 168th h. Meanwhile, T-E5 reached biomasses of 0.307 and 0.673 g L1 at the 168th h in CK and T, respectively (Fig. 2B). Though the biomass of TE5 was low in both CK and T, the T-E5 biomass in T was approximately twice that in CK, and this difference was significant. 3.4. Identification of organic acids in the root exudates by HPLC and LC/ESI-MS To determine and confirm the presence of organic acids in the concentrated root exudates, samples were subjected to HPLCcoupled LC/ESI-MS analysis. Different profiles and multiple peaks were detected in the chromatogram results of the two root exudates (Fig. 3). According to the retention times of the six organic acid standards (Fig. 3A), there were no qualitative differences between CK (Figs. 3B) and T (Fig. 3C). Three organic acids (oxalic acid, malic acid and citric acid) were identified from both CK and T. All
Fig. 3. The chromatograms of the organic acids in standard chemicals and root exudates: 1, oxalic acid; 2, malic acid; 3, acetic acid; 4, citric acid; 5, succinic acid; 6, fumaric acid. A) The chromatogram of standard organic acids. B, C) The chromatogram of the root exudates from the CK (B) and T (C) treatment groups. Notes: In split-root hydroponic system: CK, (non-inoculation, compartment 1) þ (non-inoculation, compartment 2); T, (non-inoculation, compartment 1) þ (inoculated with T. harzianum T-E5 microconidia, compartment 2).
corresponding peaks with retention times of 6.627, 8.378 and 14.820 were collected for confirmation by LC/ESI-MS. The data from LC/ESI-MS analysis are shown in Table 2. The mass spectra of peak 1 demonstrated a molecular mass of 89.1 Da [MH] in the negativeion mode; thus, peak 1 was identified as oxalic acid with a molecular weight of 90.04 Da. Similarly, the spectra of peak 2 showed a m/z ratio of 133.1 Da in the negative-ion mode and a molecular mass of 156.8 Da [M þ Na]þ under the positive-ion mode condition. The spectra of 4 exhibited a molecular mass of 214.8 Da [M þ Na]þ in the positive-ion mode. Therefore, peaks 2 and 4 were identified as malic acid and citric acid with molecular weights of 134.09 and 192.14 Da, respectively. The results overlapped with the HPLC analysis, and the collected root exudates had three organic acids in both the CK and T treatments. 3.5. Quantification of organic acids in the cucumber root exudates Fig. 2. (A) Dynamic growth of T. harzianum T-E5 in organic acid solutions 0e168 h after inoculation; (B) Biomass of T. harzianum T-E5 at 168th h. Notes: In split-root hydroponic system: CK, (non-inoculation, compartment 1) þ (non-inoculation, compartment 2); T, (non-inoculation, compartment 1) þ (inoculated with T. harzianum T-E5 microconidia, compartment 2).
The three organic acids identified were quantified by HPLC. As shown in Table 3, the amount of oxalic acid was the highest among the three organic acids. Moreover, the amount of released oxalic
F. Zhang et al. / Plant Physiology and Biochemistry 83 (2014) 250e257
255
Table 2 The liquid chromatography/electrospray ionization-mass spectrometry (LC/ESIMS) analysis of the peaks shown in Fig. 3B, C. Peak number
Retention time
Productions Negative
1 2 4
6.627 8.378 14.820
[M H] [M H]
m/z Positive [M þ Na]þ [M þ Na]þ
acid in T was approximately equivalent to 3 times that released in CK. The malic acid present in T was significantly increased by 73.03% compared with that in CK. However, there was no significant difference in citric acid amounts between CK and T. 3.6. Effects of organic acids on mycelial growth and conidial germination of Trichoderma harzianum T-E5 in vitro Exogenous applications of the previously detected organic acids (oxalic acid, malic acid and citric acid) exhibited different but significant stimulation effects on the mycelial growth of T-E5 (Fig. 4A). For oxalic acid, there was no significant difference between the stimulation rates (13.5e19.7%) at different concentrations (25e100 mM), and the colony diameter of T-E5 reached its greatest value of 7.10 cm under a 75 mM oxalic acid concentration. The T-E5 colony diameter reached its maximum value of 7.33 cm at 100 mM malic acid concentration. Moreover, the promotion percentage under 100 mM (23.6%) was significantly (P 0.05) higher than those at concentrations ranging from 25 to 75 mM (9.6e17.4%). Citric acid had greatest promotion effect (21.9%) and largest colony diameter (T-E5, 7.23 cm) at a concentration of 100 mM. Furthermore, it had the same promotion tendency with malic acid that the promotion percentage under 100 mM (21.9%) was significantly (P 0.05) higher than those at concentrations ranging from 25 to 75 mM (7.9e14.7%). In comparison with the control (0 mM), applied exogenous three organic acids significantly promoted conidial germination of T-E5 at any given concentration (25e100 mM) (Fig. 4B). Moreover, the conidial germination of T-E5 was more sensitive than its mycelial growth to the concentrations (25, 50, 75, 100 mM) of organic acids. In different oxalic acid treatments, the numbers of germinated T-E5 spores were significantly higher than the control, with stimulations ranging from 79.8% to 136.8% under concentrations ranging from 25 to 100 mM. The significant stimulation rates of malic and citric acid also increased gradually with increasing concentration until 100 mM, which generated the greatest promotion effects (161.4% in malic acid and 140.3% in citric acid) on T-E5 conidial germination.
Negative 89.1 133.1
Molecular weight
Compound
90.04 134.09 192.14
Oxalic acid Malic acid Citric acid
Positive 156.8 214.8
harzianum T-E5 colonization. The population density of T-E5 colonized on the cucumber root surface or the inner root tissues were recorded by dilution plate counting at 24, 48, 72, 96 and 120 h after inoculation. As shown in Table 4, all 3 organic acids significantly stimulated the colonization of T. harzianum T-E5 compared with the control (0 mM) at any sampling time. Specifically, oxalic acid exhibited a common promotion effect, and no significant difference was found among different concentrations ranging from 25 to 100 mM at any time point. For citric acid, the dynamic tendency of the T-E5 numbers was the same as those induced by oxalic acid (24e96 h). Subsequently, the colonization number of T-E5 at a concentration of 100 mM (11.77 104 cfu g1) was noticeably increased compared with those at 25e75 mM concentrations. Malic acid exhibited the most positive effect on the colonization of T-E5 among the 3 organic acids. When the concentration of malic acid was 100 mM, the population of T-E5 was significantly higher than those at concentrations of 25e75 mM at the 96th and 120th h. 4. Discussion In this study, we present the results of in vitro and in plant experiments regarding plant growth, fungal colonization and population dynamics, and chemical analysis. The results showed
3.7. Quantification of Trichoderma harzianum T-E5 in cucumber roots management with different organic acids A hydroponic experiment was performed to evaluate the positive effects of previously detected organic acids on Trichoderma Table 3 The quantitative determination of organic acids in cucumber root exudates by HPLC. Treatments
CK T
Organic acids (mg.g-1 root DW) Oxalic acid
Malic acid
Citric acid
1.81 ± 0.17b 4.78 ± 0.22a
0.89 ± 0.07b 1.54 ± 0.08a
0.96 ± 0.12a 1.11 ± 0.17a
Notes: In split-root hydroponic system: CK, (non-inoculation, compartment 1) þ (non-inoculation, compartment 2); T, (non-inoculation, compartment 1) þ (inoculated with T. harzianum T-E5 microconidia, compartment 2). All values are the means of three replicates. Values with different letters within the same column are significantly different at P 0.05 according to Duncan's test. Numbers followed by “±”are the standard errors (SEs).
Fig. 4. Effects of exogenous organic acid treatments on (A) mycelial growth and (B) conidial germination of T. harzianum T-E5. Notes: In split-root hydroponic system: CK, (non-inoculation, compartment 1) þ (non-inoculation, compartment 2); T, (noninoculation, compartment 1) þ (inoculated with T. harzianum T-E5 microconidia, compartment 2). Data were analyzed by Duncan's ANOVA test. Error bars represent the standard deviation of three replicates.
256
F. Zhang et al. / Plant Physiology and Biochemistry 83 (2014) 250e257
Table 4 T. harzianum T-E5 population in cucumber roots under different organic acid treatments 24e120 h after inoculation. Treatments
Concentration (mM)
104 T. harzianum cfu g1 root dry weight 24 h
Oxalic acid
Malic acid
Citric acid
0 25 50 75 100 0 25 50 75 100 0 25 50 75 100
1.36 2.68 2.79 2.91 2.89 1.36 2.24 2.38 2.42 2.49 1.36 2.37 2.46 2.45 2.59
48 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.28b 0.26a 0.22a 0.33a 0.31a 0.28b 0.32a 0.27a 0.29a 0.36a 0.28b 0.19a 0.34a 0.26a 0.29a
1.75 3.77 3.98 4.11 4.23 1.75 3.31 3.58 3.66 3.89 1.75 3.47 3.65 3.75 3.97
72 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.35b 0.36a 0.42a 0.53a 0.51a 0.35b 0.29a 0.31a 0.35a 0.42a 0.35b 0.32a 0.39a 0.33a 0.41a
2.69 4.89 5.47 6.46 6.72 2.69 4.56 4.97 5.92 6.86 2.69 4.31 4.78 5.46 6.44
96 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.29b 0.57a 0.62a 0.72a 0.69a 0.29b 0.52a 0.59a 0.63a 0.71a 0.29b 0.49a 0.55a 0.61a 0.68a
3.57 5.71 6.55 7.93 8.66 3.77 5.33 5.94 7.36 9.64 3.77 5.21 5.91 7.22 8.76
120 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.33b 0.64a 0.69a 0.82a 0.91a 0.33c 0.57b 0.68b 0.81b 0.94a 0.33b 0.59a 0.66a 0.79a 0.89a
4.29 6.76 7.87 9.46 10.56 4.59 6.27 6.81 8.56 13.06 4.59 6.03 6.89 8.75 11.77
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.51b 0.72a 0.89a 1.03a 0.98a 0.51c 0.67b 0.70b 0.93b 1.31a 0.51d 0.62c 0.72c 0.93b 1.04a
Notes: All values are the means of three replicates. Values with a different letter within the same column are significantly different at P 0.05 according to Duncan's test. Numbers followed by “±”are the standard errors (SEs).
that three organic acids (oxalic acid, malic acid and citric acid) facilitated the Trichoderma harzianum T-E5 colonization of cucumber roots. The inoculation of Trichoderma harzianum T-E5 significantly increased the biomass (shoot height and dry weights) of the cucumber plants compared with the control (CK) in the split-root hydroponic system (Table 1), which was consistent with the previous result of Yedidia et al. (2001). The report of Shivanna et al. (2005) showed that the promotion effect of Trichoderma depends on many elements, and the root colonization ability is recognized as one of the most important characteristics. Harman et al. (2004) also demonstrated that colonization by Trichoderma is beneficial to plants growth. Thus, we further observed the colonization status of T. harzianum T-E5 on the cucumber root surface by SEM (Figs. 1E, F). The hyphae of T-E5 densely covered the cucumber roots. Trichoderma spp. could efficiently colonize the root surface and, subsequently, on the elongation zone of the roots (Harman, 2000). Certain compounds in the root exudates could provide an important nutritional resource for the growth of Trichoderma to facilitate its root colonization (Vargas et al., 2009). A split-root hydroponic experiment based on plants systemic induction was designed for the collecting of cucumber root exudates in our research. Systemic induction of plant actions by microorganisms was also reported in the study of Scheffknecht et al. (2006). The cucumber root exudate solutions in compartment 1 of both CK and T were collected separately for organic acids analysis. Trichoderma harzianum T-E5 grew more rapidly and produced more mycelium in the organic acid solution collected from T than those found in CK (Fig. 2). This result indicates that T organic acid solution might provide more nutrient sources utilized by T. harzianum T-E5 to support its growth. Therefore, we further determined the compositions of the two organic acids solutions collected, and the presence of three organic acids (oxalic acid, malic acid and citric acid) were confirmed by HPLC and LC/ESI-MS analyses (Fig. 3, Table 2). Moreover, the identified organic acids were quantified in the CK and T solutions, and the results demonstrated that the secreted organic acid levels in T were significantly higher than those in CK. This result well explained the above result that the T-E5 population in T was significantly higher than in CK (Fig. 2). Many reports provide evidence to highlight the role of organic acids, noting that the overproduction of citric and malic acids can cause an increased colonization of mycorrhizal fungi and rhizobacteria in tobacco and alfalfa plants (Tesfaye et al., 2003). To our knowledge, few previous reports have demonstrated that organic acids in cucumber root exudates can function as carbon sources to
attract the colonization of Trichoderma on cucumber roots. The results obtained in vitro indicated that the three organic acids had different degrees of promotional effects on the mycelial growth and conidial germination of T-E5. Our result was consistent with those of Leonian and Lilly (1940) and showed that organic acids could effectively promote the growth of fungi. Nelson et al. (1988) also suggested that the biocontrol effect of Trichoderma koningii on Pythium seed rot mainly resulted from the addition of organic acids that stimulated the growth of Trichoderma strains. However, the reported stimulation effects were not absolutely positive for all concentrations of organic acids. In the present study, the mycelial growth and conidial germination of T. harzianum T-E5 reached their maxima when the concentrations of malic and citric acids were both 100 mM, which essentially showed that the high concentrations of organic acids provided more carbon sources for T-E5 utilization. For oxalic acid, T. harzianum T-E5 was more efficient at a concentration of 75 mM, although 100 mM offered a greater carbon source. Morton and Macmillan (1954) explained that the promotion differences mainly occurred because organic acids reduced the pH of the culture medium. T. harzianum T-E5 was most likely more sensitive to the acidity of oxalic acid than the other two organic acids (malic acid and citric acid). In other words, both the carbon source and medium pH had important influences on the growth of strain T-E5 under oxalic acid treatment. The influences of different organic acids on the dynamic colonization numbers of Trichoderma harzianum T-E5 in cucumber roots were also determined in a hydroponic system. The colonization populations of T-E5 gradually increased, along with the increased organic acid concentrations and extended culture times (Table 4). In this relatively open hydroponic environment, the influence of the surrounding acidity was less significant than nutrient supplement on the colonization number of T-E5 in cucumber roots. Moreover, the chosen culture time was appropriate because no significant decline was observed in the T-E5 population during the culture process, ensuring an accurate investigation of colonization in cucumber roots. 5. Conclusion Beneficial Trichoderma harzianum T-E5 had potential promoting effects for cucumber plant growth in split-root hydroponic system, mainly due to their efficient colonization on the cucumber root surface. The amounts of organic acids in T were significantly higher than in CK. The present study revealed that there was a strong correlation between organic acid utilization and root colonization.
F. Zhang et al. / Plant Physiology and Biochemistry 83 (2014) 250e257
After a series of in vitro and hydroponic system experiments, organic acids were proven to be used as nutritional sources by Trichoderma harzianum T-E5 to contribute to fungal population colonization on cucumber roots. Acknowledgements This work was financially supported by Innovative Research Team Development Plan of the Ministry of Education of China (IRT1256), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, the 111 project (B12009), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the Chinese Ministry of Science and Technology (2011BAD11B03), the Chinese Ministry of Agriculture (201103004). Contributions The contribution of the paper: Illustration the role of organic acids in root exudates could be performed as carbon sources to attract the colonization of Trichoderma on cucumber roots. Author Contributions: Conceived and designed the experiments: WR, FZ, QS. Performed the experiments: FZ, XM, XY. Analyzed the data: FZ. Contributed reagents/materials/analysis tools: WR, QS. Wrote the paper: FZ, WR. This research is an important and original work. Trichoderma species are well-known by their abilities of promoting plants growth and effective biocontrol of various soil-borne diseases. However, the survival rate and population density of Trichoderma colonized in the rhizosphere are the prerequisites for their effectiveness or specific functions in practice. To our knowledge, little research works had been developed to unravel the role of organic acids in root exudates could be performed as carbon sources to attract the colonization of Trichoderma on cucumber roots. We confirmed the organic acids could be used as nutritional basis of Trichoderma spp. to stimulate its colonization populations on cucumber roots in our manuscript carefully. Thus, the manuscript is an original contribution to this area. The paper may be of particular interest to the readers of your journal. References €m, S., 2000. Root-colonizing fungi from oilseed rape and their inhibition of Alstro Verticillium dahliae. J. Phytopathol. 148, 417e423. Badri, D., Vivanco, J.M., 2009. Regulation and function of root exudates. Plant. Cell. Environ. 32, 666e681. Bae, H., Sicher, R.C., Kim, M.S., Kim, S.H., Strem, M.D., Melnick, R.L., Bailey, B.A., 2009. The beneficial endophyte Trichoderma hamatum isolate DIS 219b promotes growth and delays the onset of the drought response in Theobroma cacao. J. Exp. Bot. 60, 3279e3295. Bailey, B.A., Bae, H., Strem, M.D., Roberts, D.P., Thomas, S.E., Crozier, J., Samuels, G.J., Choi, I.Y., Holmes, K.A., 2006. Fungal and plant gene expression during the colonization of cacao seedlings by endophytic isolates of four Trichoderma species. Planta 224, 1449e1464. Brotman, Y., Landau, U., Cuadros-Inostroza, A., Takayuki, T., Fernie, A.R., Chet, I., Viterbo, A., Willmitzer, L., 2013. Trichoderma-plant root colonization: escaping early plant defense responses and activation of the antioxidant machinery for saline stress tolerance. Plos. Pathog. 9. Chen, L.H., Huang, X.Q., Zhang, F.G., Zhao, D.K., Yang, X.M., Shen, Q.R., 2012. Application of Trichoderma harzianum SQR-T037 bio-organic fertiliser significantly controls Fusarium wilt and affects the microbial communities of continuously cropped soil of cucumber. J. Sci. Food. Agr. 92. s-Penagos, C., Lo pez-Bucio, J., Contreras-Cornejo, H.A., Macías-Rodríguez, L.I., Corte 2009. Trichoderma virens, a plant beneficial fungus, enhances biomass production and promotes lateral root growth through an auxin-dependent mechanism in Arabidopsis. Plant. Physiol. 149, 1579e1592. Elad, Y., Chet, I., Henis, Y., 1981. A selective medium for improving quantitative isolation of Trichoderma spp. from soil. Phyloparasitica 9, 59e67. Hao, W.Y., Ren, L.X., Ran, W., Shen, Q.R., 2010. Allelopathic effects of root exudates from watermelon and rice plants on Fusarium oxysporum f.sp. niveum. Plant. Soil. 336, 485e497. Harman, G.E., 2000. Combining effective strains of Trichoderma harzianum and solid matrix priming to improve biological seed treatments. Plant. Dis. 84, 377e393.
257
Harman, G.E., Howell, C.R., Viterbo, A., Chet, I., Lorito, M., 2004. Trichoderma speciesoportunistic, avirulent plant symbionts. Nat. Rev. Microbiol. 2, 43e56. Hiltner, L., 1904. Ueber neuere Erfahrungen und Probleme auf dem Gebiete der Bodenbakteriologie und unter besonderer BerUcksichtigung der Grundungung und Brache. Arb. Deut. Landw. Gesell 98, 59e78. Howell, C.R., 2002. Cotton seedling preemergence damping-off incited by Rhizopus oryzae and Pythium spp and its biological control with Trichoderma spp. Phytopathology 92, 177e180. Hoyos-Carvajal, L., Orduz, S., Bissett, J., 2009. Growth stimulation in bean (Phaseolus vulgaris L.) by Trichoderma. Biocontrol 51, 409e416. Huang, X.Q., Chen, L.H., Ran, W., Shen, Q.R., Yang, X.M., 2011. Trichoderma harzianum strain SQR-T37 and its bio-organic fertilizer could control Rhizoctonia solani damping-off disease in cucumber seedlings mainly by the mycoparasitism. Appl. Microbiol. Biot. 91, 741e755. Leonian, L.H., Lilly, V.G., 1940. Studies on the nutrition of fungi. IV. Factors influencing the growth of some thiamine-requiring fungi. Am. J. Bot. 27, 18e26. Lilia, C.C., Paul, G.D., Dmitri, F., Mohammad-Reza, H., Rainer, B., Nicolaus, V.W., 2011. Root exudation of sugars, amino acids, and organic acids by maize as affected by nitrogen, phosphorus, potassium, and iron deficiency. J. Plant. Nutr. Soil. Sci. 174, 3e11. Ling, N., Raza, W., Ma, J.H., Huang, Q.W., Shen, Q.R., 2011. Identification and role of organic acids in watermelon root exudates for recruiting Paenibacillus polymyxa SQR-21 in the rhizosphere. Eur. J. Soil. Biol. 47, 374e379. Ling, N., Huang, Q.W., Guo, S.W., Shen, Q.R., 2011. Paenibacillus polymyxa SQR-21 systemically affects root exudates of watermelon to decrease the conidial germination of Fusarium oxysporum f. sp. niveum. Plant. Soil. 341, 485e493. Liu, F., Wen, X.S., 2006. Progress in relationship between root exudates and rhizospheric microorganism. Food. Drug. 8, 37e40. pez-Bucio, J., Nieto-Jacobo, M.F., Ramírez-Rodríguez, V., Herrera-Estrella, L., 2000. Lo Organic acid metabolism in plants: from adaptive physiology to transgenic varieties for cultivation in extreme soils. Plant. Sci. 160, 1e13. Mogan, J.A.W., Bending, G.D., White, P.J., 2005. Biological costs and benefits to plant-microbe interactions in the rhizosphere. J. Exp. Bot. 56, 729e1739. Morton, A.G., Macmillan, A., 1954. The assimilation of nitrogen from ammonium salts and nitrate by fungi. J. Exp. Bot. 5, 232e252. Nelson, E.B., Harman, G.E., Nash, G.T., 1988. Enhancement of Trichoderma-induced biological control of pythium seed rot and pre-emergence dramping-off of peas. Soil. Biol. Biochem 20, 145e150. Raaijmakers, J.M., Paulitz, T.C., Steinberg, C., Alabouvette, C., Moenne-Loccoz, Y., 2009. The rhizosphere: a playground and battlefield for soilborne pathogens and beneficial microorganisms. Plant. Soil. 321, 341e361. Rabeendran, N., Jones, E.E., Moot, D.J., Stewart, A., 2006. Biocontrol of Sclerotinia lettuce drop by Coniothyrium minitans and Trichoderma hamatum. Biol. Control 39, 352e362. , L.B., 2009. The role of microbial Randy, O.C., Hexon, A.C.C., Lourdes, M.R., Jose signals in plant growth and development. Plant. Signal. Behav. 4, 701e712. Rigot, J., Matsumura, F., 2002. Assessment of the rhizosphere competency and pentachlorophenol- metabolizing activity of a pesticide-degrading strain of Trichoderma harzianum introduced into the root zone of corn seedlings. J. Environ. Sci. Heal. B 37, 201e210. Scheffknecht, S., Mammerler, R., Steinkellner, S., Vierheilig, H., 2006. Root exudates of mycorrhizal tomato plants exhibit a different effect on microconidia germination of Fusarium oxysporum f. sp. lycopersici than root exudates from nonmycorrhizal tomato plants. Mycorrhiza 16, 365e370. Shivanna, M.B., Meera, M.S., Kubota, M., Hyakumachi, M., 2005. Promotion of growth and yield in cucumber by zoysiagrass rhizosphere fungi. Microbes. Environ. 2, 34e40. Swarnalee, D., Rani, T.S., Appa, R.P., 2013. Root exudate-Induced alterations in Bacillus cereus cell wall contribute to root colonization and plant growth promotion. Plos. One. http://dx.doi.org/10.1371/journal.pone.0078369. Tan, S.Y., Yang, C.L., Mei, X.L., Shen, S., Raza, W., Shen, Q.R., Xu, Y.C., 2013. The effect of organic acids from tomato root exudates on rhizosphere colonization of Bacillus amyloliquefaciens T-5. Appl. Soil. Ecol. 64, 15e22. Tesfaye, M., Dufault, N.S., Dornbusch, M., Allan, D., Vance, C.P., Samac, D.A., 2003. Influence of enhanced malate dehydrogenase expression by alfalfa on diversity of rhizobacteria and soil nutrient availability. Soil. Biol. Biochem 35, 1103e1113. Vargas, W.A., Mandawe, J.C., Kenerley, C.M., 2009. Plant-derived sucrose is a key element in the symbiotic association between Trichoderma virens and maize plants. Plant. Physiol. 151, 792e808. , Y., 2003. Systemic inhibition of arbuscular mycorrhiza Vierheilig, H., Lerat, S., Piche development by root exudates of cucumber plants colonized by Glomus mosseae. Mycorrhiza 13, 167e170. Yedidia, I., Srivastva, A.K., Kapulnik, Y., Chet, I., 2001. Effect of Trichoderma harzianum on microelement concentrations and increased growth of cucumber plants. Plant. Soil. 235, 235e242. Zhang, F.G., Zhu, Z., Yang, X.M., Ran, W., Shen, Q.R., 2013. Trichoderma harzianum TE5 significantly affects cucumber root exudates and fungal community in the cucumber rhizosphere. Appl. Soil. Ecol. 72, 41e48. Zhang, F.G., Yuan, J., Yang, X.M., Cui, Y.Q., Chen, L.H., Ran, W., Shen, Q.R., 2013. Putative Trichoderma harzianum mutant promotes cucumber growth by enhanced production of indole acetic acid and plant colonization. Plant. Soil. 368, 433e444. Zhang, F.G., Zhu, Z., Wang, B.B., Wang, P., Yu, G.H., Wu, M.J., Chen, W., Ran, W., Shen, Q.R., 2013. Optimization of Trichoderma harzianum T-E5 biomass and determining the degradation sequence of biopolymers by FTIR in solid-state fermentation. Ind. Crop. Prod. 49, 619e627.
