Effect of Elevated Atmospheric CO2 and Temperature on the Disease Severity of Rocket Plants Caused by Fusarium Wilt under Phytotron Conditions

Walter Chitarra; Ilenia Siciliano; Ilario Ferrocino; Maria Lodovica Gullino; Angelo Garibaldi

doi:10.1371/journal.pone.0140769

Abstract

The severity of F. oxysporum f.sp. conglutinans on rocket plants grown under simulated climate change conditions has been studied. The rocket plants were cultivated on an infested substrate (4 log CFU g^-1) and a non-infested substrate over three cycles. Pots were placed in six phytotrons in order to simulate different environmental conditions: 1) 400–450 ppm CO₂, 18–22°C; 2) 800–850 ppm CO₂, 18–22°C; 3) 400–450 ppm CO₂, 22–26°C, 4) 800–850 ppm CO₂, 22–26°C, 5) 400–450 ppm CO₂, 26–30°C; 6) 800–850 ppm CO₂, 26–30°C. Substrates from the infested and control samples were collected from each phytotron at 0, 60 and 120 days after transplanting. The disease index, microbial abundance, leaf physiological performances, root exudates and variability in the fungal profiles were monitored. The disease index was found to be significantly influenced by higher levels of temperature and CO₂. Plate counts showed that fungal and bacterial development was not affected by the different CO₂ and temperature levels, but a significant decreasing trend was observed from 0 up to 120 days. Conversely, the F. oxysporum f.sp. conglutinans plate counts did not show any significantly decrease from 0 up to 120 days. The fungal profiles, evaluated by means of polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE), showed a relationship to temperature and CO₂ on fungal diversity profiles. Different exudation patterns were observed when the controls and infested plants were compared, and it was found that both CO₂ and temperature can influence the release of compounds from the roots of rocket plants. In short, the results show that global climate changes could influence disease incidence, probably through plant-mediated effects, caused by soilborne pathogens.

Citation: Chitarra W, Siciliano I, Ferrocino I, Gullino ML, Garibaldi A (2015) Effect of Elevated Atmospheric CO₂ and Temperature on the Disease Severity of Rocket Plants Caused by Fusarium Wilt under Phytotron Conditions. PLoS ONE 10(10): e0140769. https://doi.org/10.1371/journal.pone.0140769

Editor: Vijai Gupta, National University of Ireland - Galway, IRELAND

Received: July 31, 2015; Accepted: September 30, 2015; Published: October 15, 2015

Copyright: © 2015 Chitarra et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper and its Supporting Information file.

Funding: This research was supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634179 "Effective Management of Pests and Harmful Alien Species - Integrated Solutions" (EMPHASIS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Nowadays, how climate changes will influence plant-pathogen interactions and their impact on production is largely debated and represents a challenge for future programmes focused on disease management under global change conditions [1]. An increasing numbers of multidisciplinary approaches have reported the effects of rising temperature and CO₂ levels on crop productions and physiological changes [2]. On the other hand, researchers have only recently focused on plant disease prediction and management under climate changes [3]. Plant pathogens represents an important constraint for the security of our future food as a consequence of population increases, urbanization, globalization and changes in climate [4]. Moreover, several pathogens produce toxins and other compounds that are dangerous to human and animal health which could affect market and world trade. On these grounds, a multidisciplinary approach is needed to implement predictive models and include higher levels of ecological interactions [5]. As recently reported by an Intergovernmental Panel on Climate Changes [6], anthropogenic emissions of greenhouse gases have caused negative impacts on human and natural systems over the last three decades. In other words, changes in the atmospheric composition and temperature as well as humidity alterations can have an impact on host plant physiology and influence a plant’s resistance against pathogens [7–9]. On one hand, rises in CO₂ and temperature can affect the sense and responses of a plant by increasing the photosynthesis rates, water and light-use efficiency, leaf surface properties, changes in anatomy, morphology, phenology and root exudates, thus profoundly modifying native plants and soil microbial communities [1,3,8,10,11]. On the other hand, elevated CO₂ influences the pathogenicity, host-pathogen interaction and epidemiology of fungal diseases [3,12–15,16]. An enlarged canopy coupled with a favourable microclimate offers more sites for infection and increases fungal pathogen fecundity, which has been shown to lead to twice the number of lesions for high CO₂ concentrations [17,18]. Furthermore, the mutation and selection of plant pathogens and the consequent development of new strains have also been predicted [3,19].

Phytotron-based studies are optimized to study the interactions that occur between plants, soil and soil microbiota and they combine environmental parameters such as light intensity, CO₂ concentrations, temperature, and relative humidity [20]. Phytotron chambers have in fact been largely used to study the effects of global changes on several pathosystems [11, 14–16,21]. Plants directly or indirectly affect the development of soil microbiota in the rhizosphere and in bulk soil by means of root exudates [22]. However, most studies conducted so far report contrasting results; such as a decrease [23], an increase [24] or no changes [25] in microbial diversity and activities. The development of soil microbial populations needs a multiphasic approach that can couple traditional microbial analysis with molecular tools such as polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). This technique has been optimized to monitor soil microflora changes under climate change conditions [16,26,27]. Fusarium oxysporum and the related formae specialis cause greater economic damage to several crops than other plant pathogens [28]. Rocket (Eruca sativa) is a high-value horticultural crop, mainly cultivated in the Mediterranean area, up to 3–5 times in the same soil which is affected by emerging soilborne pathogens [20,29] and Fusarium oxysporum f.sp. conglutinans has recently been detected in Italy on cultivated (Eruca sativa) and wild rocket (Diplotaxis tenuifolia) [30].

The aim of this work was to study the effect of F. oxysporum f.sp. conglutinans, artificially infested in a growing substrate, on rocket plants grown under simulated climate change conditions with rising CO₂ concentrations and temperatures in phytotron chambers. Six temperature/CO₂ combinations were studied. Disease incidence, the physiological performances of leaves, microbial and fungal cultivable abundance and the main root exudate components were monitored to evaluate the effects of climatic changes on disease development over time. Furthermore, shifts in fungal communities were assessed using the DGGE technique on DNA directly extracted from an infested growing substrate.

Materials and Methods

Inoculum preparation

F. oxysporum f.sp. conglutinans (ATCC16600RB, Agroinnova, Grugliasco, Italy), which is resistant to benomyl [31], was used and cultured in 1000-mL Erlenmeyer flasks containing 250 mL of hydrolized casein. The flasks were incubated on a platform shaker at 20–25°C for 12 days. Chlamydospores were recovered by means of centrifugation for 20 min at 8000g at 20°C, following the removal of mycelia fragments by sieving through cheesecloth. The chlamydospore suspension was dried and mixed with sterile talc powder (1:2 w/w), as described by Locke & Colhoun [32], and stored at room temperature for further use. The number of chlamydospores per gram of talc was assessed by serial plating on a Komada medium [33] containing 10 mg L^-1 of benomyl (Benlate, 50% a.i.; DuPont de Nemours, Milan, Italy). The talc formulation was incorporated into the soil to achieve the desired inoculum of 4 Log colony forming units (CFU) g^-1.

Plant material and experimental set-up

Two experimental trials were carried out at Agroinnova (Grugliasco, Itay). Plastic tanks containing 100L of a mixture (1:1 v/v) of peat-perlite substrate (Tecno2, Turco Silvestro sphagnum peat moss, Albenga, SV, Italy) and sandy loam soil (pH, 7.3; organic matter content, 2.2%; cation exchange capacity, 2.6 meq/100 g soil) were prepared. The final substrate was made up of: sand, 76%; silt, 14%; clay, 10%; pH, 7.51; organic matter content, 2,59% and had a cation exchange capacity of 5.99 meq/100 g soil and subjected to steam sterilization before use. The substrate was artificially inoculated with F. oxysporum f. sp. conglutinans (ATCC16600RB) to reach a final concentration of 4 Log CFU ml^-1. A non-inoculated tank was used as the control.

Eruca sativa Mill seeds (cultivated rocket) were disinfected in a solution of 1% sodium hypocloride plus 0.01% of Tween-20, and then rinsed twice in water for 1 min. The seeds were air-dried at RT and stored at 4°C until use. The seeds were sown in a greenhouse in plug trays (20–26°C, 70% RH and natural light condition). After 15–20 days, the first seedling-leaves were developed. The rocket plants were left to grow under Phytotron conditions for 7 days. Subsequently, 48 pots (2L each) were prepared from the inoculated tank and another 48 pots were prepared from the non-inoculated tank and used as controls. The rocket plants were then transplanted (4 plants/pot), and 8 inoculated and 8 non-inoculated pots were placed in 6 different phytotrons (PGC 9.2, TECNO.EL, Italy). One replicate consisted of two pots. The rocket plants were kept in the phytotrons under six different temperature and CO₂ combinations according to the following ranges: 1) 400–450 ppm CO₂, 18–22°C; 2) 800–850 ppm CO₂, 18–22°C; 3) 400–450 ppm CO₂, 22–26°C, 4) 800–850 ppm CO₂, 22–26°C, 5) 400–450 ppm CO₂, 26–30°C; 6) 800–850 ppm CO₂, 26–30°C. The temperatures, light and humidity were changed gradually during the day in order to simulate natural conditions. Three subsequent cultivation cycles were carried out in the same pot. Each crop cycle lasted 35–37 days after transplanting. The plants were irrigated daily in order to maintain the soil moisture at field capacity.

Substrate samples were taken at time 0 (immediately before plant transplanting) and after 60 and 120 days for microbial enumeration and molecular analyses (PCR-DGGE), while the physiological measurements of the plant leaves were conducted at the end of each rocket cycle.

Disease incidence evaluation

The effectiveness of the different simulated climate change conditions on the severity of F. oxysporum f.sp. conglutinans on rocket was checked weekly by evaluating the pathogen development using a previously reported disease index [34]. Wilted plants were counted and removed and the final disease rating was made at the end of the experiment (35–37 days after transplanting). At the end of each cycle, re-isolation from infected plants on a Komada medium supplemented with 10 mg L^-1 of benomyl was performed to confirm the presence of F. oxysporum as the causal agent of the observed symptoms. During the latter survey, the total fresh plant biomass was also rated using a technical balance (Orma SNC, Milano, Italy) to evaluate the effect of the treatments on plant growth.

Physiological measurements of the leaves

In order to observe the effects of the climate change conditions on the leaf physiological activity of the infected and control rocket plants, the photosynthetic efficiency and chlorophyll content were monitored. Measurements were performed following the experimental protocol reported by Pugliese et al. [35], with only minor modifications.

The chlorophyll content index (CCI) was measured with the SPAD 502 chlorophyll meter (CCM-200, Opti-Sciences, Inc., Hudson, NH, USA), which determined the relative amount of chlorophyll in the leaf by measuring the absorbance in the red and near-infrared regions (650 and 940 nm, respectively). Chlorophyll meter readings were taken from each rocket plant in the second or third leaves (fully developed) from the top on ten randomly selected plants (one leaf/plant) at the end of each cultivation cycle.

The photosynthetic efficiency measurements were performed on five randomly selected leaves using a portable continuous-excitation type fluorimeter (Handy-PEA, Hansatech Instruments Ltd, Norfolk, UK), according to the manufacturer’s instructions, at the end of each cultivation cycle of rocket plants grown in infested and control substrates.

Root exudate analyses

In order to investigate the relationship between climate changes and the turnover of low molecular weight organic compounds in the rhizosphere, water-soluble root exudates were collected and analyzed. A sterilized CaSO₄ 0.01M solution (collection media) was used to collect the root exudates [36]. For each condition tested, three randomly selected rocket plants were taken from infested and not infested pots and the soil was carefully removed using deionized water, without damaging the roots. The plants were then placed in a 50 mL centrifuge tube with 15 mL of collection media for 2 hours in each phytotron. The collection media was then filtered through a 0.45 μm membrane filter. pH was measured and the samples were freeze dried and dissolved in 3 mL of ddH₂O. Three main groups of organic substances were analyzed: total organic carbon (TOC), total organic acids and amino acid compounds.

TOC was determined by means of the colorimetric method. An aliquot of 1 mL was mixed with 2 mL of K₂Cr₂O₇ (2 N) and 1 mL of H₂SO₄ concentrate and kept at 150°C for 10 min. The samples were cooled to room temperature and then analyzed by using a Spectroquant Pharo 300 spectrophotometer (Merck, Darmstadt, Germany) at λ = 585 nm. Glucose was used as the reference C substance. For the amino acids analysis, 1 mL of sample was mixed with 1 mL of borate buffer (0.4 M pH = 9.5) and 1 mL of dansyl chloride solution (Sigma-Aldrich, St. Louis, MO, USA) at 10 mg/mL. The samples were incubated at 65°C for 30 min [37]. The analysis was performed using an Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA). Zorbax Eclipse Plus C18 (4.6x100 mm, 3.5 μm, Agilent Technologies) was used for the chromatographic separation of the amino acids with a linear gradient from 10 to 100% of acetonitrile (Merck) in 15 min. The derivatized amino acids were detected by means of a fluorescent detector with excitation at 335 nm and emission at 524 nm. A mixture of 20 standard amino acids (Sigma-Aldrich) was used as the reference. The organic acids were separated with a Synergi Hydro-RP column (4.6x250 mm, 4 μm, Phenomenex, Torrance, CA, USA) in an isocratic condition, using buffer phosphate (50 mM) as the mobile phase. The detector was set at λ = 214 nm. A mixture of standard organic acids (Sigma-Aldrich) was used as the reference.

Microbial count

Substrate samples (25 grams) were collected from 8 pots for both infested and control treatments, at a depth of 2–5 cm, and were placed in sterilized polyethylene bags using a sterilized spatula. The samples were passed through a sieve to remove any vegetation, and mixed at room temperature for 30 min with 225 mL of quarter strength Ringer’s solution (Merck) in sterilized flasks on a rotary shaker (100 rpm). Decimal dilutions were prepared in quarter strength Ringer’s solution and aliquots of 1 ml of the appropriate dilutions were poured, in triplicate, onto the following media: plate count agar (PCA, Oxoid, Milan) incubated at 28°C for 48h for the total bacterial counts; potato dextrose agar (PDA, Merck) supplemented with streptomycin (0,5 g/L) and a Komada medium supplemented with 10 mg L^-1 of benomyl (Benlate, 50% w.g., DuPont, USA), kept at 25°C for 7 days for the total fungal and F. oxysporum f.sp. conglutinans enumerations, respectively. The results were calculated as the mean of the Log counts of three independent determinations.

Substrate DNA extraction and DGGE analysis

Infested substrate samples (250 mg) were taken from 4 pots/phytotron at 0, 60 and 120 days after transplanting for DNA extraction. DNA exctract according to the protocol described by the NucleoSpin® Soil manufacturer (Macherey-Nagel, Germany) and was quantified using the NanoDrop 1000 spectrophotometer (Thermo Scientific) and standardized at 10 ng μl^-1. Fungal DNA was amplified using fungal ITS primers according to Gao et al. [38]. PCR products for the ITS region were analyzed by denaturing gradient gel electrophoresis (DGGE) at 25–35% using a Bio-Rad Dcode, as previously described [16].

Statistical analysis

The data obtained from the plate counts, root exudates and leaf physiological measurements were analyzed using one-way analysis of variance (ANOVA), with treatments being the main factor. ANOVA analyses were performed with the SPSS 22.0 statistical software package (SPSS Inc., Cary, NC, USA). The Duncan HSD test was applied when ANOVA revealed significant differences (P < 0.05). A database of fingerprints was created using the PyElph software. A combined data matrix that included all the fingerprints for the ITS region was obtained, and dendrograms of similarity were retrieved using the Dice coefficient and the Unweighted Pair Group Method with the Arithmetic Average (UPGMA) clustering algorithm [39] utilizing PyElph software [40]. The similarity distance matrix generated through PyElph was used to build a Projection on Latent Structures—Discriminant Analysis (PLS-DA) utilizing “mixOmics” in the R environment (www.r-project.org). The obtained binary band-matching tables were considered to calculate the Shannon-Wiener diversity index (H’) [41] using PAST (PAleontological STatistics) software [42].

Results

Disease index assessment and fresh biomass production

The disease index (DI, 0–100) was found to be significantly influenced by the temperature and CO₂ levels (Table 1). No symptoms were observed for the control plants in any of the cycles or phytotron conditions that were considered. Generally, higher levels of CO₂ and temperature were factors that significantly influenced the DI in the infected plants in the simulated conditions. At 18–22°C, the DI was significantly lower for both CO₂ ranges (P < 0.05) than the other conditions. Conversely, the DI significantly increased at 22–26°C, particularly for higher CO₂ levels (P < 0.05) where a DI of 54 was reached. At 26–30°C, a slightly decreasing trend was recorded for both CO₂ levels, although a higher DI (43,4) was observed at 800–850 ppm CO_2, with a significantly different value than that of the 18–22°C conditions (P < 0.05). The fresh weight (FW) data pertaining to the infected plants showed significant differences (P < 0.05) as a consequence of temperature, CO₂ and disease development (Table 1). Lower FW values were obtained at 18–22°C for both CO₂ conditions and in phytotrons 4 and 6 (800–850 ppm CO₂, 22–26°C and 800–850 ppm CO₂, 26–30°C, respectively) where disease development was higher (Table 1). The control plants showed a higher FW than the diseased rocket plants, with the exception of 18–22°C (phytotrons 1 and 2: 400–450 ppm CO₂, 18–22°C and 800–850 ppm CO₂, 18–22°C) in which FW was significantly lower (P < 0.05) than the other tested.

Download:

Table 1. Disease index (0–100) and total fresh weight of the plants at the end of the replicates (FW).

https://doi.org/10.1371/journal.pone.0140769.t001

Effect on the physiological performances of the leaves

The photosynthetic efficiency index (PI) for the infected plants showed a particularly pronounced and statistically significant (P < 0.05) decreasing trend for all the conditions tested at the end of the cycles (Fig 1). In other words, PI appeared to be particularly affected by disease development (Table 1) coupled with the environmental conditions. Lower PI values (P < 0.05) were recorded for the infected plants from phytotrons 1, 2 and 4 (400–450 ppm CO₂, 18–22°C; 800–850 ppm CO₂, 18–22°C and 800–850 ppm CO₂, 22–26°C, respectively). Moreover, the highest temperature provided the best evidence of impaired PI at high CO₂ in the infected plants.

Download:

Fig 1. Photosynthetic efficiency measurements.

Effect of different CO₂ and temperature combinations on the photosynthetic efficiency of the leaves (PI) of rocket plants grown in a substrate artificially infested with F. oxysporum f.sp. conglutinans and the control. Tukey's HSD test (P < 0.05).

https://doi.org/10.1371/journal.pone.0140769.g001

In general, the non-inoculated plants showed higher photosynthetic efficiency than the infected ones, with the exception of those from phytotron 4 (800–850 ppm CO₂, 22–26°C) where PI was lower than for the other conditions.

Similar trends were also observed for the CCI measurements in the infected samples (Fig 2) where no significant differences were observed between the phytotrons (P < 0.05). The non-infected plants showed higher levels of chlorophyll content than the infected rocket plants. Furthermore, significantly lower values were measured in the non-infected plants in phytotrons 3 and 5 (400–450 ppm CO₂, 22–26°C and 400–450 ppm CO₂, 26–30°C, respectively) (P < 0.05). Overall, the physiological measurements have confirmed that the disease development led to a remarkable decrease in the physiological performances of the leaves.

Download:

Fig 2. Assessment of chlorophyll content.

Effect of different CO₂ and temperature combinations on the chlorophyll content of the leaves (CCI, °SPAD) of rocket plants grown in a substrate artificially infested with F. oxysporum f.sp. conglutinans and the control. Tukey's HSD test (P < 0.05).

https://doi.org/10.1371/journal.pone.0140769.g002

Effect on root exudates

The results pertaining to the root exudates (Table 2) showed a similar trend for TOC and amino acids (AA), when the exudates from the infected roots and the control plants were compared. In both cases, when the temperature was lower (18–22°C), an increased concentration was registered in the samples from the infected plants. At 22–26°C and 26–30°C, the TOC and AA concentrations significantly increased for higher CO₂ levels (P < 0.05). No significant differences were observed between the infected and non-infected plants for lower CO₂ concentrations. The root exudates from the infected samples did not show any significant differences in TOC concentration at 400–450 ppm of CO₂; conversely, an increasing trend of TOC concentration was registered when the temperature and CO₂ level were increased (P < 0.05).

Download:

Table 2. Main root exudate components analyzed on rocket plants cultivated in infested and control substrates collected at the end of the cycle.

https://doi.org/10.1371/journal.pone.0140769.t002

The amino acid levels from the infected plants were not different at lower CO₂ levels; conversely, higher CO₂ levels only induced a significant increase in amino acids at high temperatures (P < 0.05).

The organic acids, in general, showed a different trend. When CO₂ and temperature were kept low, no significant differences were registered between the treatments or in the phytotrons. However, increasing the concentration of CO₂ led to an increased production of organic acids, but only in the control samples. Furthermore, at 22–26°C, for both levels of CO₂, the organic acid concentration increased for all the samples. In general, the organic acid concentrations of all the samples were always significantly higher for higher CO₂ levels (P < 0.05).

Microbial analysis of substrate samples

The results of the bacterial and fungal plate counts on specific media are shown in Tables 3, 4 and 5. The plate counts of mesophilic bacteria (TBC) (Table 3) from infested substrate samples were not affected by the different CO₂ and temperature levels. A significant decreasing trend was observed from time 0 up to 120 days for all the conditions considered (P < 0.05). Furthermore, the samples at 120 days were not significantly different between the phytotrons. The control substrate samples showed a similar trend to that observed in the infested samples. However, the samples taken after 120 days in phytotrons 2 and 4 (800–850 ppm CO₂, 18–22°C and 800–850 ppm CO₂, 22–26°C) were significantly higher (P < 0.05), that is, 7.17 and 6.87 Log CFU g^-1, respectively.

Download:

Table 3. Total bacterial counts (TBC) of mesophilic bacteria from the infested and control substrate samples following incubation in phytotrons for 120 days.

https://doi.org/10.1371/journal.pone.0140769.t003

Download:

Table 4. Total fungi community counts (TFC) from the infested and control substrate samples following incubation in phytotrons for 120 days.

https://doi.org/10.1371/journal.pone.0140769.t004

Download:

Table 5. Plate counts of Fusarium oxysporum f.sp. conglutinans from the infested substrate samples following incubation in phytotrons for 120 days.

https://doi.org/10.1371/journal.pone.0140769.t005

The total fungal community plate counts (TFC, Table 4) from the infested samples mirrored the bacterial counts; a significant decreasing trend (P < 0.05) was observed over time from time 0, without any significant differences between the phytotrons at 120 days. The control substrate sample counts were similar to the infested ones, with the exception of those from phytotron 2 (18–22°C and 850 ppm CO₂) at 120 days, when the statistical values were higher (4.89 Log CFU g^-1) than in the other conditions. However, (among fungal community) Fusarium oxysporum f.sp. conglutinans development was observed over time on the Komada medium supplemented with benomyl in the infested substrate samples. In general, most of the fungal community was represented by the pathogen, which developed over time and almost completely covered the total fungal community values up to the last survey (120 days). In particular, a general increasing trend was observed for all the conditions from time 0 in the phytotrons compared to time 120. Although no significant differences between phytotrons were observed at 120 days. A significant increasing trend from time 0 was observed for phytotrons 2 and 4 for high CO₂ conditions in the last survey (from 3.80 to 4.38 and from 3.73 to 4.32 Log CFU g^-1, respectively) (P < 0.05). With the exception of 26–30°C, high concentrations of CO₂ were shown to positively affect the growth of Fusarium oxysporum f.sp. conglutinans. No pathogen was detected in the control substrate samples.

DGGE analysis of the fungal microbiota

The PCR-DGGE fingerprints of the fungal community obtained from the DNA extracted directly from the infested samples in all the adopted conditions are presented in Fig 3. Repeated DNA extraction and PCR-DGGE analysis confirmed the results of the fingerprinting.

Download:

Fig 3. PLS-DA models based on DGGE similarity distance matrix.

Plot A, PLS-DA models based on the DGGE similarity matrix as a function of CO₂: 400–450 ppm (blue), 800–850 ppm (yellow); Plot B, PLS-DA models based on the DGGE similarity matrix as a function of the temperature: 26°C (blue), 22°C (yellow), and 30°C (red): Plot C, PLS-DA models based on the DGGE similarity matrix as a function of the time: time 0 (red), time 60 (yellow) and time120 (blue); Plot D; PLS-DA models based on the DGGE similarity matrix as a function of the phytotrons: 1 (red), 2 (yellow), 3 (blue), 4 (black), 5 (green), 6 (pink).

https://doi.org/10.1371/journal.pone.0140769.g003

The ITS based DGGE analysis showed an average result of 11.7 bands per sample (min 3, max 17), (S1 Fig). The similarity matrix generated through the PyElph software was used to build a PLS-DA, as a function of the CO₂ concentration, and the results showed a clear separation between samples at 850 and samples at 450 ppm CO₂ (Fig 3A). Regardless of the temperature, the PLS models showed a clear separation of the samples (Fig 3B). When the samples were grouped on the basis of the sampling time, the samples at time 0 were clearly separated from the other samples (Fig 3C). The PLS-DA analysis, as a function of the phytotrons (Fig 3D), resulted in a certain degree of separation. The separation was particularly important for the samples from phytotron 4, which appear to group together and to be separated from the other phytotrons.

Furthermore, a binary band-matching table was analyzed in order to calculate the index of diversity (Shannon-Wiener diversity index H’). The results, using only the data from the ITS region, revealed, on the basis of their phytotron origin, that there was a biological diversity in the samples. In particular, the H’ index and richness of the fungal community changed according to the environmental conditions that were set, and ranged from 1.61 to 2.83 and 5 to 17, respectively. At the end of the experiment, the H’ index (1.79) and richness (10) were lower for phytotrons 4 and 6 (22–26 and 26–30°C at 850 ppm CO₂), and the H’ index (2.83) and richness (17) were higher for Phytotron 1 (18–22°C and 450 ppm CO₂). Higher CO₂ and temperature were shown to be selective and to reduce fungal community diversity.

Discussion

Plants that grow under elevated temperature and CO₂ concentrations often exhibit positive responses by increasing photosynthesis and/or water and nutrient use efficiency [43]. However, how increases in CO₂ might affect plant-pathogen interaction has not been addressed or debated to any great extent. The aim of this work was to evaluate the effect of simulated climate changes, by increasing CO₂ concentrations and temperature levels, on the severity of Fusarium oxysporum f.sp. conglutinans on rocket plants. Phytotron chambers have already been used in several studies, since they provide total control of the environmental conditions and reproducible data in diverse pathosystems [20, 14–16]. Climate change is an on-going phenomenon, which affects whole ecosystems and causes multitrophic interactions that are difficult to understand [1]. The possibility of reproducing different climatic conditions by coupling carbon dioxide and temperature levels can/has been considered be very useful to obtain a better understanding of plant-pathogen interactions, as has been done in the present work which deals with a soilborne pathogen. Focusing on plant-soil systems, it is well known that plants directly or indirectly control and influence the multitrophic interactions of the soil through root exudates [22]. The results reported in this study refer to disease incidence, cultivable abundance, leaf physiological performances, root exudate analyses, and variability in substrate fungal profiles by PCR-DGGE. Several studies have reported that rising CO₂ concentrations can cause plants to modify their root architecture and exudation compounds in the rhizosphere [44,45]. In addition, it has been predicted that global warming will directly influence multitrophic interactions if the soil temperature exceeds its buffering capacity and this could be followed by changes in the quality, quantity and diversity of plant and soil microbial communities and therefore plant pathogen development [1]. In the present study, a good disease level was reached under artificial inoculation of F. oxysporum f.sp. conglutinans, thus making it possible to evaluate the effect of the different climate change simulations. The temperatures and CO₂ combinations in the six environmental conditions had a significant influence on the incidence of Fusarium wilt. Higher CO₂ concentrations and temperatures caused a significant increase in DI, in particular in the 22–26°C range (Phytotron 4, Table 1). The effect of increased CO₂ concentrations was still observed at 26–30°C on disease severity development, although lower values were observed than in the 22–26°C range, probably due to the suboptimal range of temperature that limits pathogen development. Although elevated CO₂ had a significant effect on Fusarium wilt incidence, the increase in temperature also had a significant effect on disease development, particularly at 22–26°C, which is a favourable range of temperatures for disease development [30]. As previously reported, higher temperature or CO₂ levels generally correspond to a greater severity of Fusarium wilt in other crops such as lettuce [16,46] or wheat [47]. As reviewed by Ainsworth and Rogers [8], rising CO₂ concentrations and temperatures could influence leaf physiology, morphology and indirectly crop production by increasing or blocking their metabolic performances and photosynthetic efficiency. However, no relevant effects linked to disease development or environment conditions have been observed on PI and CCI data for infested and control plants, respectively. In fact, in infected rocket plants, large reductions of both indices have been recorded compared to controls, thus suggesting the high susceptibility of the photosynthetic machinery already at lower disease severity values. Furthermore, photosynthetic efficiency and leaf chlorophyll content are indicators of photosynthetic activity and chlorophyll stability. Fluorimeters and SPAD chlorophyll meters are frequently used for the measurement of foliar damage provoked by different biotic and abiotic stresses [35,48], although they were not considered useful for the present investigation.

Recent research in plant biology has pointed out the importance and the role of root exudates in mediating biological interactions in the rhizosphere. The chemical components of root exudates could deter or attract an organism with different effects on the plants. This would be particularly important during the pathogenesis of root-infecting fungal pathogens. However, the signalling and composition of root exudates in plant-pathogen interactions have not yet been elucidated [49–51]. To the best of the authors’ knowledge, this is the first report to have been made on the analysis of root exudates on wilted rocket plants under simulated climate change conditions. The results have shown a different exudation pattern between the controls and infected plants. Interestingly, higher levels of pH, organic acid and TOC (mainly composed of sugars) were observed in the infected samples in phytotron 4 where the disease index was higher. In addition, high values of organic acid, TOC and amino acids were also recorded in phytotron 6, where a consistent level of DI was observed. These results suggest the combined temperature and high CO₂ effect on root component release. In particular, the TOC compounds underwent a significant increase, since they represent a carbon source for microbial metabolism and energy that in turn could influence the attraction and severity of the soilborne pathogen. Similarly, Kerks et al. [52] reported the activation of root exudate chemotaxis and pathogenicity genes of S. enterica serovar Typhimurium in lettuce, which are involved in root attachment and subsequent colonization. Although a great deal of information is available concerning the relationships between symbionts and plants, limited knowledge exists about the communication between plants and root pathogens mediated by rhizodeposits and/or by pathogen metabolite production (e.g. volatile organic compounds, VOCs) [53]. Further studies are needed to increase analytical skills and to analyse the chemistry of root exudates, and thus to resolve the dialogue that takes place between pathogens and plant roots. In addition, root exudates represent the main source of soil organic carbon, defined as soluble low-molecular weight components that are mainly composed of sugars, amino acids, organic acids and other secondary metabolites which vary from plant to plant and which are able to shape the rhizosphere microbiome [22]. Conversely, no relevant effects of CO₂ or temperature levels have been observed in the control plants to explain the variation in the root exudate components in phytotron conditions, in line with what has been reported by Uselman et al. [54] in Robinia pseudoacacia.

It is well known that DGGE fingerprints can be used to describe microbial composition and diversity, but they do not provide any information on the abundance and concentration of separate microbial species [55]. For this reason, this limitation has partially been addressed here by conducting plate counts on several media. In the present experimental conditions, the total bacteria and fungi plate counts in both the infested and control substrate samples showed a decreasing trend over time up to the end of the experiment, without climate simulation effects being observed. These results suggested that the forecast increase in CO₂ and temperature levels due to climate changes would have a limited effect on fungal and bacterial development. Recent reports [16,26,56] have shown similar results, while other studies have reported results that depend to a great extent upon the host, microorganisms and environment [57]. Conversely, the F. oxysporum f.sp. conglutinans plate counts at the end of the present experiment have shown a slight increase or no significant variation for time 0, except for phytotron 4 at higher CO₂ concentrations, where the development and disease incidence were significantly higher. In phytotron 6, where disease incidence was high, no significant development of pathogen over time was detected. This behaviour supports the hypothesis of a plant-mediated effect increasing disease incidence. The variability in the fungal profiles was assessed by means of the PCR-DGGE approach, which has frequently been used, with good results, in other simulated global change studies [16,26]. The samples were clearly separated in relation to the temperature or CO₂. In addition, this result was further confirmed from an examination of the Shannon-Wiener indices. Interestingly, phytotrons 4 and 6 showed the least diversity and the highest DI. Thus, a low diversity might have a higher impact on disease development efficiency than the species visualized from the DGGE examination. The loss in diversity caused by higher temperature and CO₂ levels could be due to a plant-mediated effect that causes the selection of a few dominant species and which may exclude others species through a competition strategy.

Conclusions

The disease incidence of pathogenic Fusarium species could increase due to the effects of the global changes that have been predicted for the future. The present experimental conditions have shown a coupled temperature and CO₂ effect on disease severity, which is probably plant-mediated, as reported for other pathosystems. Although no specific predictions can be made on field conditions, the data obtained from phytotron growth chambers could help to unravel the complexity of plant-soilborne pathogen interactions that take place under climate change conditions, in order to implement model prediction and prevention strategies.

Supporting Information

S1 Fig. Similarity dendrogram generated from the digitized PCR-DGGE fingerprints of DNA directly extracted from the substrate infested with F. oxysporum f.sp. conglutinans.

https://doi.org/10.1371/journal.pone.0140769.s001

(TIF)

Acknowledgments

This research was supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634179 "Effective Management of Pests and Harmful Alien Species—Integrated Solutions" (EMPHASIS). The authors are grateful to Marguerite Jones for the English editing.

Author Contributions

Conceived and designed the experiments: AG MLG WC. Performed the experiments: WC IS. Analyzed the data: WC IS IF. Contributed reagents/materials/analysis tools: AG MLG. Wrote the paper: WC IS IF AG MLG.

References

1. Chakraborty S, Pangga IB, Roper MM. Climate change and multitrophic interaction in soil: the primacy of plants and functional domains. Glob Chang Biol. 2012; 18:2111–2125.
- View Article
- Google Scholar
2. Lobell DB, Gourdji SM. The influence of climate change on global crop productivity. Plant Physiol. 2012; 160(4):1686–1697. pmid:23054565
- View Article
- PubMed/NCBI
- Google Scholar
3. Chakraborty S. Migrate or evolve: options for plant pathogens under climate change. Glob Chang Biol. 2013; 19:1985–2000. pmid:23554235
- View Article
- PubMed/NCBI
- Google Scholar
4. Gregory PJ, Jhonson SN, Newton AC, Ingram JSI. Integrating pests and pathogens into the climate change/food security debate. J Exp Bot. 2009; 60:2827–2838. pmid:19380424
- View Article
- PubMed/NCBI
- Google Scholar
5. Shabani F, Kumar L, Esmaeili A. Future distribution of Fusarium oxysporum f. spp. in European, Middle Eastern and North African agricultural regions under climate change. Agric Ecosist Environ. 2014; 197:96–105.
- View Article
- Google Scholar
6. IPCC Intergovernmental Panel on Climate Change. IPCC. 2013. Fifth Assessment Report: Summary for Policymakers. 36p.
7. Pangga IB, Chakraborty S, Yates D. Canopy Size and induced resistance in Stylosanthes scabra determine anthracnose severity at High CO₂. Phytopathol. 2004; 94(3):221–227.
- View Article
- Google Scholar
8. Ainsworth E, Rogers A. The response of photosynthesis and stomatal conductance to rising [CO₂]: mechanisms and environmental interactions. Plant Cell Environ. 2007; 30:258–270. pmid:17263773
- View Article
- PubMed/NCBI
- Google Scholar
9. Lake J, Wade R. Plant-pathogen interactions and elevated CO2: morphological changes in favour of pathogens. J Exp Bot. 2009; 60:3123–3131. pmid:19470658
- View Article
- PubMed/NCBI
- Google Scholar
10. Harley SE, Jones CJ, Couper GC, Jones TH. Biosynthesis of plant phenolic compounds in elevated atmospheric CO₂. Glob Chang Biol. 2000; 6:497–506.
- View Article
- Google Scholar
11. Chakraborty S. Potential impact of climate change on plant-pathogen interactions. Australas Plant Pathol. 2005; 34:443–448.
- View Article
- Google Scholar
12. Mithcell CE, Reich PB, Tilman D, Groth JV. Effects of elevated CO₂, nitrogen deposition, and decreased species diversity on foliar fungal plant disease. Glob Chang Biol. 2003; 9:438–451.
- View Article
- Google Scholar
13. Eastburn DM, Degennaro MM, Delucia EH, Dermody O, Mcelrone AJ. Elevated atmospheric carbon dioxide and ozone alter soybean diseases at SoyFACE. Glob Chang Biol. 2010; 16:320–330.
- View Article
- Google Scholar
14. Pugliese M, Cogliati E, Gullino ML, Garibaldi A. Effect of climate change on Alternaria leaf spot of rocket salad and black spot of basil under controlled environment. Commun Agric Appl Biol Sci. 2012a; 77: 241–4.
- View Article
- Google Scholar
15. Pugliese M, Liu J, Titone P, Garibaldi A, Gullino ML. Effect of elevated CO₂ and temperature on interactions of zucchini and powdery mildew. Phytopathol Mediterr. 2012b; 51: 480–487.
- View Article
- Google Scholar
16. Ferrocino I, Chitarra W, Pugliese M, Gilardi G, Gulino ML, Garibaldi A. Effect of elevated atmospheric CO₂ and temperature on disease severity of Fusarium oxysporum f.sp. lactucae on lettuce plants. Appl Soil Ecol. 2013; 72:1–6.
- View Article
- Google Scholar
17. Chakraborty S, Pangga IB, Lupton J, Hart L, Room PM, Yates D. Production and dispersal of Colletotrichum gleosporoides spores on Stylosanthes scabra under elevated CO₂. Environ Poll. 2000; 108:381–387.
- View Article
- Google Scholar
18. Pangga IB. Effects of elevated CO₂ on plant architecture of Stylosanthes scabra and epidemiology of anthracnose disease. PhD Thesis, University of Queensland. 2002.
19. Chakraborty S, Datta S. How will plant pathogens adapt to host plant resistance at elevated CO₂ under a changing climate?. New Phytol. 2003; 159:733–742.
- View Article
- Google Scholar
20. Gullino ML, Garibaldi A. Soil solarization under greenhouse conditions. In: Gamliel A, Katan J. editors. Soil solarization: Theory and Practice. St. Paul, MN, USA, APS Press; 2012. pp 187–191.
21. Ainsworth EA, Long SP. What have we learned from 15 years of free-air CO2 enrichment (FACE)? A meta-analytic review of the responses of photosynthesis, canopy properties and plant production to rising CO2. New Phytol. 2005; 165(2):351–71. pmid:15720649
- View Article
- PubMed/NCBI
- Google Scholar
22. Haichar FZ, Santaella C, Heulin T, Achouak W. Root exudates mediated interactions belowground. Soil Biol Biochem. 2014; 77:69–80.
- View Article
- Google Scholar
23. Ebersberger D, Niklaus PA, Kandeler E. Long term CO2 enrichment stimulates N-mineralisation and enzyme activities on calcareous grassland. Soil Biol Biochem. 2003; 35:965–972.
- View Article
- Google Scholar
24. Cheng L, Booker FL, Burkey KO, Tu C, Shew HD. Soil microbial responses to elevated CO₂ and O₃ in a nitrogen-aggrading agroecosystem. PLoS One. 2011; 6:e21377. pmid:21731722
- View Article
- PubMed/NCBI
- Google Scholar
25. Zack DR, Pregitzer KS, Curtis PS, Holmes WE. Atmospheric CO2 and the composition and function of soil microbial communities. Ecol Appl. 2000; 10:47–59.
- View Article
- Google Scholar
26. Guenet B, Lenhart K, Leloup J, Giusti-Miller S, Pouteau V, Mora P, et al. The impact of long-term CO₂ enrichment and moisture levels on soil microbial community structure and enzyme activities. Geoderma. 2012; 170:331–336.
- View Article
- Google Scholar
27. Ferrocino I, Gilardi G, Pugliese M, Gullino ML, Garibaldi A. Shifts in ascomycete community of biosolarized substrate infested with Fusarium oxysporum f.sp. conglutinans and F. oxysporum f.sp. basilici by PCR-DGGE. Appl Soil Ecol. 2014; 81:12–21.
- View Article
- Google Scholar
28. Correll J. The relationship between formae speciales, races, and vegetative compatibility groups in Fusarium oxysporum. Phytopathology. 1991; 81:1061–1064.
- View Article
- Google Scholar
29. Garibaldi A, Gullino ML. Emerging soilborne diseases of horticultural crops and new trends in their management. Acta Hortic. 2010; 883:37–46.
- View Article
- Google Scholar
30. Garibaldi A, Gilardi G, Gullino ML. Evidence for an expanded host range of Fusarium oxysporum f.sp. raphani. Phytoparassitica. 2006; 34:115–121.
- View Article
- Google Scholar
31. Lu P, Ricauda Aimonino D, Gilardi G, Gullino ML, Garibaldi A. Efficacy of different steam distribution systems against five soilborne pathogens under controlled laboratory conditions. Phytoparassitica. 2010; 38:175–189.
- View Article
- Google Scholar
32. Locke T, Colhoun J. Contributions to a method of testing oil palm seedlings for resistance to Fusarium oxysporum Schl. f. sp. elaeidis Toovey. Phytopathololy. 1974; 79:77–92.
- View Article
- Google Scholar
33. Komada H. Development of selective medium for quantitative isolation of Fusarium oxysporum from natural soil. Rev Plant Prot Res. 1975; 8:114–125.
- View Article
- Google Scholar
34. Gilardi G, Demarchi S, Gullino ML, Garibaldi A. Effect of simulated soil solarization and organic amendments on Fusarium wilt of rocket and basil under controlled conditions. J Phytopathol. 2013; 162: 557–566.
- View Article
- Google Scholar
35. Pugliese M, Gullino ML, Garibaldi A. Effects of elevated CO₂ and temperature on interactions of grapevine and powdery mildew: first results under phytotron conditions. J Plant Dis Prot. 2010; 117: 9–14.
- View Article
- Google Scholar
36. Aulakh MS, Wassmann R, Bueno C, Kreuzwieser J, Rennenberg H. Characterization of root exudates at different growth stages of ten rice (Oryza sativa L.) cultivar. Plant Biol. 2001; 3:139–148.
- View Article
- Google Scholar
37. Mazzotti F, Benabdelkamel H, Di Donna L, Athanassopoulos CM, Napoli A, Sindona G. Light and heavy dansyl reporter groups in food chemistry: amino acid assay in beverages. J Mass Spectrom. 2012; 47:932–939. pmid:22791261
- View Article
- PubMed/NCBI
- Google Scholar
38. Gao G, Yin D, Chen S, Xia F, Yang J, Li K, et al. Effect of biocontrol agent Pseudomonas fluorescens 2P24 on soil fungal community in cucumber rhizosphere using T-RFLP and DGGE. PLoS One. 2012; 7(2):e31806. pmid:22359632
- View Article
- PubMed/NCBI
- Google Scholar
39. Vauterin L, Vauterin P. Computer-aided objective comparison of electrophoretic patterns for grouping and identification of microorganisms. Eur. Microbiol. 1992; 1:37–41.
- View Article
- Google Scholar
40. Pavel BA, Vasile IC. PyElph—a software tool for gel images analysis and phylogenetics. BMC Bioinformatics. 2012; 13:9. pmid:22244131
- View Article
- PubMed/NCBI
- Google Scholar
41. Shannon AE, Weaver W. (1949) The Mathematical Theory of Communities. University Illinois Press: 1949; Urbana, IL, USA.
42. Hammer Ø, Harper DAT, Ryan PD. Past: paleontological statistics software package for education and data analysis. Palaeontologia Electron. 2001;4:9–18.
- View Article
- Google Scholar
43. Kimball BA, Kobayashi K, Bindi M. Responses of agricultural crops to free-air CO₂ enrichment. Adv Agron. 2002; 77:293–368.
- View Article
- Google Scholar
44. Pritchard SG. Soil organisms and global climate change. Plant Pathol. 2011; 60:82–99.
- View Article
- Google Scholar
45. Pickles BJ, Genney DR, Anderson IC, Alexander IJ. Spatial analysis of ectomycorrhizal fungi reveals that root tip communities are structured by competitive interactions. Mol Ecol. 2012; 21(20):5510–5123.
- View Article
- Google Scholar
46. Scott JC, Gordon TR. Effect of Temperature on Severity of Fusarium Wilt of Lettuce Caused by Fusarium oxysporum f. sp. lactucae. Plant Dis. 2010; 94:13–17.
- View Article
- Google Scholar
47. Melloy P, Hollaway G, Luck J, Norton R, Aitken E, Chakraborty S. Production and fitness of Fusarium pseudograminearum inoculum at elevated carbon dioxide in FACE. Global Chang Biol. 2010; 16(12): 3363–337.
- View Article
- Google Scholar
48. Bijanzadeh E, Emam Y. Effect of defoliation and drought stress on yield components and chlorophyll content of wheat. Pak J Biol Sci. 2010; 13:699–705. pmid:21848062
- View Article
- PubMed/NCBI
- Google Scholar
49. Bais HP, Weir TL, Perry LG, Gilroy S, Vivanco JM. The role of root exudates in rhizosphere interactions with plants and other organisms. Annu Rev Plant Biol. 2006; 57:233–266. pmid:16669762
- View Article
- PubMed/NCBI
- Google Scholar
50. Berendsen RL, Pieterse CMJ, Bakker PAHM. The rhizosphere microbiome and plant health. Trends Plant Sci. 2012; 17:478–486. pmid:22564542
- View Article
- PubMed/NCBI
- Google Scholar
51. Piattoni F, Roberti R, Servidio G, D’Aulerio AZ. Studies on the potential role of root exudates in the interaction between musk melon roots and Fusarium oxysporum f.sp. melonis. J Plant Dis Prot. 2014; 121(2):64–70.
- View Article
- Google Scholar
52. Kerks MM, Franz E, Gent-Pelzer M, Zijlstra C, Bruggen AHC. Differential interaction of Salmonella enterica serovars with lettuce cultivars and plant-microbe factors influencing the colonization efficiency. ISME J. 2007; 1:620–631. pmid:18043669
- View Article
- PubMed/NCBI
- Google Scholar
53. Mendes R, Garbeva P, Raaijmakers JM. The rhizosphere microbiome: significance of plant beneficial, plant pathogenic, and human pathogenic microorganisms. FEMS Microbiol Rev. 2013; 37:634–663. pmid:23790204
- View Article
- PubMed/NCBI
- Google Scholar
54. Uselman SM, Qualls RG, Thomas RB. Effects of increased atmospheric CO₂, temperature, and soil N availability on root exudation of dissolved organic carbon by a N-fixing tree (Robinia pseudoacacia L.). Plant Soil. 2000; 222:191–202.
- View Article
- Google Scholar
55. Gafan G, Lucas V, Roberts G. Statistical analyses of complex denaturing gradient gel electrophoresis profiles. J Clin Microbiol. 2005; 43:3971–3978. pmid:16081938
- View Article
- PubMed/NCBI
- Google Scholar
56. Runion GB, Prior SA, Price AJ, McElroy JS, Torbert HA. Effects of elevated CO₂ on biomass and fungi associated with two ecotypes of ragweed (Ambrosia artemisiifolia L.). Front Plant Sci. 2014; 5:500. pmid:25309569
- View Article
- PubMed/NCBI
- Google Scholar
57. Ziska LH, Runion GB. Future weed, pest, and disease problems for plants. In: Carran RA, Edwards GR, Niklaus PS, editors. Agroecosystem in a Changing Climate. Newton PCD, Boca Raton, FL: CRC Press; 2007. pp. 261–287.

[ref1] 1. Chakraborty S, Pangga IB, Roper MM. Climate change and multitrophic interaction in soil: the primacy of plants and functional domains. Glob Chang Biol. 2012; 18:2111–2125.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Lobell DB, Gourdji SM. The influence of climate change on global crop productivity. Plant Physiol. 2012; 160(4):1686–1697. pmid:23054565
View Article
PubMed/NCBI
Google Scholar

[5] View Article

[6] PubMed/NCBI

[7] Google Scholar

[ref3] 3. Chakraborty S. Migrate or evolve: options for plant pathogens under climate change. Glob Chang Biol. 2013; 19:1985–2000. pmid:23554235
View Article
PubMed/NCBI
Google Scholar

[9] View Article

[10] PubMed/NCBI

[11] Google Scholar

[ref4] 4. Gregory PJ, Jhonson SN, Newton AC, Ingram JSI. Integrating pests and pathogens into the climate change/food security debate. J Exp Bot. 2009; 60:2827–2838. pmid:19380424
View Article
PubMed/NCBI
Google Scholar

[13] View Article

[14] PubMed/NCBI

[15] Google Scholar

[ref5] 5. Shabani F, Kumar L, Esmaeili A. Future distribution of Fusarium oxysporum f. spp. in European, Middle Eastern and North African agricultural regions under climate change. Agric Ecosist Environ. 2014; 197:96–105.
View Article
Google Scholar

[17] View Article

[18] Google Scholar

[ref6] 6. IPCC Intergovernmental Panel on Climate Change. IPCC. 2013. Fifth Assessment Report: Summary for Policymakers. 36p.

[ref7] 7. Pangga IB, Chakraborty S, Yates D. Canopy Size and induced resistance in Stylosanthes scabra determine anthracnose severity at High CO₂. Phytopathol. 2004; 94(3):221–227.
View Article
Google Scholar

[21] View Article

[22] Google Scholar

[ref8] 8. Ainsworth E, Rogers A. The response of photosynthesis and stomatal conductance to rising [CO₂]: mechanisms and environmental interactions. Plant Cell Environ. 2007; 30:258–270. pmid:17263773
View Article
PubMed/NCBI
Google Scholar

[24] View Article

[25] PubMed/NCBI

[26] Google Scholar

[ref9] 9. Lake J, Wade R. Plant-pathogen interactions and elevated CO2: morphological changes in favour of pathogens. J Exp Bot. 2009; 60:3123–3131. pmid:19470658
View Article
PubMed/NCBI
Google Scholar

[28] View Article

[29] PubMed/NCBI

[30] Google Scholar

[ref10] 10. Harley SE, Jones CJ, Couper GC, Jones TH. Biosynthesis of plant phenolic compounds in elevated atmospheric CO₂. Glob Chang Biol. 2000; 6:497–506.
View Article
Google Scholar

[32] View Article

[33] Google Scholar

[ref11] 11. Chakraborty S. Potential impact of climate change on plant-pathogen interactions. Australas Plant Pathol. 2005; 34:443–448.
View Article
Google Scholar

[35] View Article

[36] Google Scholar

[ref12] 12. Mithcell CE, Reich PB, Tilman D, Groth JV. Effects of elevated CO₂, nitrogen deposition, and decreased species diversity on foliar fungal plant disease. Glob Chang Biol. 2003; 9:438–451.
View Article
Google Scholar

[38] View Article

[39] Google Scholar

[ref13] 13. Eastburn DM, Degennaro MM, Delucia EH, Dermody O, Mcelrone AJ. Elevated atmospheric carbon dioxide and ozone alter soybean diseases at SoyFACE. Glob Chang Biol. 2010; 16:320–330.
View Article
Google Scholar

[41] View Article

[42] Google Scholar

[ref14] 14. Pugliese M, Cogliati E, Gullino ML, Garibaldi A. Effect of climate change on Alternaria leaf spot of rocket salad and black spot of basil under controlled environment. Commun Agric Appl Biol Sci. 2012a; 77: 241–4.
View Article
Google Scholar

[44] View Article

[45] Google Scholar

[ref15] 15. Pugliese M, Liu J, Titone P, Garibaldi A, Gullino ML. Effect of elevated CO₂ and temperature on interactions of zucchini and powdery mildew. Phytopathol Mediterr. 2012b; 51: 480–487.
View Article
Google Scholar

[47] View Article

[48] Google Scholar

[ref16] 16. Ferrocino I, Chitarra W, Pugliese M, Gilardi G, Gulino ML, Garibaldi A. Effect of elevated atmospheric CO₂ and temperature on disease severity of Fusarium oxysporum f.sp. lactucae on lettuce plants. Appl Soil Ecol. 2013; 72:1–6.
View Article
Google Scholar

[50] View Article

[51] Google Scholar

[ref17] 17. Chakraborty S, Pangga IB, Lupton J, Hart L, Room PM, Yates D. Production and dispersal of Colletotrichum gleosporoides spores on Stylosanthes scabra under elevated CO₂. Environ Poll. 2000; 108:381–387.
View Article
Google Scholar

[53] View Article

[54] Google Scholar

[ref18] 18. Pangga IB. Effects of elevated CO₂ on plant architecture of Stylosanthes scabra and epidemiology of anthracnose disease. PhD Thesis, University of Queensland. 2002.

[ref19] 19. Chakraborty S, Datta S. How will plant pathogens adapt to host plant resistance at elevated CO₂ under a changing climate?. New Phytol. 2003; 159:733–742.
View Article
Google Scholar

[57] View Article

[58] Google Scholar

[ref20] 20. Gullino ML, Garibaldi A. Soil solarization under greenhouse conditions. In: Gamliel A, Katan J. editors. Soil solarization: Theory and Practice. St. Paul, MN, USA, APS Press; 2012. pp 187–191.

[ref21] 21. Ainsworth EA, Long SP. What have we learned from 15 years of free-air CO2 enrichment (FACE)? A meta-analytic review of the responses of photosynthesis, canopy properties and plant production to rising CO2. New Phytol. 2005; 165(2):351–71. pmid:15720649
View Article
PubMed/NCBI
Google Scholar

[61] View Article

[62] PubMed/NCBI

[63] Google Scholar

[ref22] 22. Haichar FZ, Santaella C, Heulin T, Achouak W. Root exudates mediated interactions belowground. Soil Biol Biochem. 2014; 77:69–80.
View Article
Google Scholar

[65] View Article

[66] Google Scholar

[ref23] 23. Ebersberger D, Niklaus PA, Kandeler E. Long term CO2 enrichment stimulates N-mineralisation and enzyme activities on calcareous grassland. Soil Biol Biochem. 2003; 35:965–972.
View Article
Google Scholar

[68] View Article

[69] Google Scholar

[ref24] 24. Cheng L, Booker FL, Burkey KO, Tu C, Shew HD. Soil microbial responses to elevated CO₂ and O₃ in a nitrogen-aggrading agroecosystem. PLoS One. 2011; 6:e21377. pmid:21731722
View Article
PubMed/NCBI
Google Scholar

[71] View Article

[72] PubMed/NCBI

[73] Google Scholar

[ref25] 25. Zack DR, Pregitzer KS, Curtis PS, Holmes WE. Atmospheric CO2 and the composition and function of soil microbial communities. Ecol Appl. 2000; 10:47–59.
View Article
Google Scholar

[75] View Article

[76] Google Scholar

[ref26] 26. Guenet B, Lenhart K, Leloup J, Giusti-Miller S, Pouteau V, Mora P, et al. The impact of long-term CO₂ enrichment and moisture levels on soil microbial community structure and enzyme activities. Geoderma. 2012; 170:331–336.
View Article
Google Scholar

[78] View Article

[79] Google Scholar

[ref27] 27. Ferrocino I, Gilardi G, Pugliese M, Gullino ML, Garibaldi A. Shifts in ascomycete community of biosolarized substrate infested with Fusarium oxysporum f.sp. conglutinans and F. oxysporum f.sp. basilici by PCR-DGGE. Appl Soil Ecol. 2014; 81:12–21.
View Article
Google Scholar

[81] View Article

[82] Google Scholar

[ref28] 28. Correll J. The relationship between formae speciales, races, and vegetative compatibility groups in Fusarium oxysporum. Phytopathology. 1991; 81:1061–1064.
View Article
Google Scholar

[84] View Article

[85] Google Scholar

[ref29] 29. Garibaldi A, Gullino ML. Emerging soilborne diseases of horticultural crops and new trends in their management. Acta Hortic. 2010; 883:37–46.
View Article
Google Scholar

[87] View Article

[88] Google Scholar

[ref30] 30. Garibaldi A, Gilardi G, Gullino ML. Evidence for an expanded host range of Fusarium oxysporum f.sp. raphani. Phytoparassitica. 2006; 34:115–121.
View Article
Google Scholar

[90] View Article

[91] Google Scholar

[ref31] 31. Lu P, Ricauda Aimonino D, Gilardi G, Gullino ML, Garibaldi A. Efficacy of different steam distribution systems against five soilborne pathogens under controlled laboratory conditions. Phytoparassitica. 2010; 38:175–189.
View Article
Google Scholar

[93] View Article

[94] Google Scholar

[ref32] 32. Locke T, Colhoun J. Contributions to a method of testing oil palm seedlings for resistance to Fusarium oxysporum Schl. f. sp. elaeidis Toovey. Phytopathololy. 1974; 79:77–92.
View Article
Google Scholar

[96] View Article

[97] Google Scholar

[ref33] 33. Komada H. Development of selective medium for quantitative isolation of Fusarium oxysporum from natural soil. Rev Plant Prot Res. 1975; 8:114–125.
View Article
Google Scholar

[99] View Article

[100] Google Scholar

[ref34] 34. Gilardi G, Demarchi S, Gullino ML, Garibaldi A. Effect of simulated soil solarization and organic amendments on Fusarium wilt of rocket and basil under controlled conditions. J Phytopathol. 2013; 162: 557–566.
View Article
Google Scholar

[102] View Article

[103] Google Scholar

[ref35] 35. Pugliese M, Gullino ML, Garibaldi A. Effects of elevated CO₂ and temperature on interactions of grapevine and powdery mildew: first results under phytotron conditions. J Plant Dis Prot. 2010; 117: 9–14.
View Article
Google Scholar

[105] View Article

[106] Google Scholar

[ref36] 36. Aulakh MS, Wassmann R, Bueno C, Kreuzwieser J, Rennenberg H. Characterization of root exudates at different growth stages of ten rice (Oryza sativa L.) cultivar. Plant Biol. 2001; 3:139–148.
View Article
Google Scholar

[108] View Article

[109] Google Scholar

[ref37] 37. Mazzotti F, Benabdelkamel H, Di Donna L, Athanassopoulos CM, Napoli A, Sindona G. Light and heavy dansyl reporter groups in food chemistry: amino acid assay in beverages. J Mass Spectrom. 2012; 47:932–939. pmid:22791261
View Article
PubMed/NCBI
Google Scholar

[111] View Article

[112] PubMed/NCBI

[113] Google Scholar

[ref38] 38. Gao G, Yin D, Chen S, Xia F, Yang J, Li K, et al. Effect of biocontrol agent Pseudomonas fluorescens 2P24 on soil fungal community in cucumber rhizosphere using T-RFLP and DGGE. PLoS One. 2012; 7(2):e31806. pmid:22359632
View Article
PubMed/NCBI
Google Scholar

[115] View Article

[116] PubMed/NCBI

[117] Google Scholar

[ref39] 39. Vauterin L, Vauterin P. Computer-aided objective comparison of electrophoretic patterns for grouping and identification of microorganisms. Eur. Microbiol. 1992; 1:37–41.
View Article
Google Scholar

[119] View Article

[120] Google Scholar

[ref40] 40. Pavel BA, Vasile IC. PyElph—a software tool for gel images analysis and phylogenetics. BMC Bioinformatics. 2012; 13:9. pmid:22244131
View Article
PubMed/NCBI
Google Scholar

[122] View Article

[123] PubMed/NCBI

[124] Google Scholar

[ref41] 41. Shannon AE, Weaver W. (1949) The Mathematical Theory of Communities. University Illinois Press: 1949; Urbana, IL, USA.

[ref42] 42. Hammer Ø, Harper DAT, Ryan PD. Past: paleontological statistics software package for education and data analysis. Palaeontologia Electron. 2001;4:9–18.
View Article
Google Scholar

[127] View Article

[128] Google Scholar

[ref43] 43. Kimball BA, Kobayashi K, Bindi M. Responses of agricultural crops to free-air CO₂ enrichment. Adv Agron. 2002; 77:293–368.
View Article
Google Scholar

[130] View Article

[131] Google Scholar

[ref44] 44. Pritchard SG. Soil organisms and global climate change. Plant Pathol. 2011; 60:82–99.
View Article
Google Scholar

[133] View Article

[134] Google Scholar

[ref45] 45. Pickles BJ, Genney DR, Anderson IC, Alexander IJ. Spatial analysis of ectomycorrhizal fungi reveals that root tip communities are structured by competitive interactions. Mol Ecol. 2012; 21(20):5510–5123.
View Article
Google Scholar

[136] View Article

[137] Google Scholar

[ref46] 46. Scott JC, Gordon TR. Effect of Temperature on Severity of Fusarium Wilt of Lettuce Caused by Fusarium oxysporum f. sp. lactucae. Plant Dis. 2010; 94:13–17.
View Article
Google Scholar

[139] View Article

[140] Google Scholar

[ref47] 47. Melloy P, Hollaway G, Luck J, Norton R, Aitken E, Chakraborty S. Production and fitness of Fusarium pseudograminearum inoculum at elevated carbon dioxide in FACE. Global Chang Biol. 2010; 16(12): 3363–337.
View Article
Google Scholar

[142] View Article

[143] Google Scholar

[ref48] 48. Bijanzadeh E, Emam Y. Effect of defoliation and drought stress on yield components and chlorophyll content of wheat. Pak J Biol Sci. 2010; 13:699–705. pmid:21848062
View Article
PubMed/NCBI
Google Scholar

[145] View Article

[146] PubMed/NCBI

[147] Google Scholar

[ref49] 49. Bais HP, Weir TL, Perry LG, Gilroy S, Vivanco JM. The role of root exudates in rhizosphere interactions with plants and other organisms. Annu Rev Plant Biol. 2006; 57:233–266. pmid:16669762
View Article
PubMed/NCBI
Google Scholar

[149] View Article

[150] PubMed/NCBI

[151] Google Scholar

[ref50] 50. Berendsen RL, Pieterse CMJ, Bakker PAHM. The rhizosphere microbiome and plant health. Trends Plant Sci. 2012; 17:478–486. pmid:22564542
View Article
PubMed/NCBI
Google Scholar

[153] View Article

[154] PubMed/NCBI

[155] Google Scholar

[ref51] 51. Piattoni F, Roberti R, Servidio G, D’Aulerio AZ. Studies on the potential role of root exudates in the interaction between musk melon roots and Fusarium oxysporum f.sp. melonis. J Plant Dis Prot. 2014; 121(2):64–70.
View Article
Google Scholar

[157] View Article

[158] Google Scholar

[ref52] 52. Kerks MM, Franz E, Gent-Pelzer M, Zijlstra C, Bruggen AHC. Differential interaction of Salmonella enterica serovars with lettuce cultivars and plant-microbe factors influencing the colonization efficiency. ISME J. 2007; 1:620–631. pmid:18043669
View Article
PubMed/NCBI
Google Scholar

[160] View Article

[161] PubMed/NCBI

[162] Google Scholar

[ref53] 53. Mendes R, Garbeva P, Raaijmakers JM. The rhizosphere microbiome: significance of plant beneficial, plant pathogenic, and human pathogenic microorganisms. FEMS Microbiol Rev. 2013; 37:634–663. pmid:23790204
View Article
PubMed/NCBI
Google Scholar

[164] View Article

[165] PubMed/NCBI

[166] Google Scholar

[ref54] 54. Uselman SM, Qualls RG, Thomas RB. Effects of increased atmospheric CO₂, temperature, and soil N availability on root exudation of dissolved organic carbon by a N-fixing tree (Robinia pseudoacacia L.). Plant Soil. 2000; 222:191–202.
View Article
Google Scholar

[168] View Article

[169] Google Scholar

[ref55] 55. Gafan G, Lucas V, Roberts G. Statistical analyses of complex denaturing gradient gel electrophoresis profiles. J Clin Microbiol. 2005; 43:3971–3978. pmid:16081938
View Article
PubMed/NCBI
Google Scholar

[171] View Article

[172] PubMed/NCBI

[173] Google Scholar

[ref56] 56. Runion GB, Prior SA, Price AJ, McElroy JS, Torbert HA. Effects of elevated CO₂ on biomass and fungi associated with two ecotypes of ragweed (Ambrosia artemisiifolia L.). Front Plant Sci. 2014; 5:500. pmid:25309569
View Article
PubMed/NCBI
Google Scholar

[175] View Article

[176] PubMed/NCBI

[177] Google Scholar

[ref57] 57. Ziska LH, Runion GB. Future weed, pest, and disease problems for plants. In: Carran RA, Edwards GR, Niklaus PS, editors. Agroecosystem in a Changing Climate. Newton PCD, Boca Raton, FL: CRC Press; 2007. pp. 261–287.

Figures

Abstract

Introduction

Materials and Methods

Inoculum preparation

Plant material and experimental set-up

Disease incidence evaluation

Physiological measurements of the leaves

Root exudate analyses

Microbial count

Substrate DNA extraction and DGGE analysis

Statistical analysis

Results

Disease index assessment and fresh biomass production

Effect on the physiological performances of the leaves

Effect on root exudates

Microbial analysis of substrate samples

DGGE analysis of the fungal microbiota

Discussion

Conclusions

Supporting Information

S1 Fig. Similarity dendrogram generated from the digitized PCR-DGGE fingerprints of DNA directly extracted from the substrate infested with F. oxysporum f.sp. conglutinans.

Acknowledgments

Author Contributions

References