NMR Analysis Reveals a Wealth of Metabolites in Root-Knot Nematode Resistant Roots of Citrullus amarus Watermelon Plants

Seeds of seven homozygous resistant germplasm lines, each derived (through self-pollination and single seed descent through four-five generations) from a PI resistant to RKN (CA) and watermelon cultivars (CL; Crimson Sweet and Charleston Gray) susceptible to RKN (Thies and Levi, 2003, 2007) were grown in a greenhouse at the USDA, ARS, U.S. Vegetable Laboratory in Charleston, SC. All seven PIs showed similar levels of resistance in the field studies performed by Thies and Levi (2007). The seeds were sowed in trays with individual cells containing a steam-sterilized Metro Mix 360 Potting Soil (Sungro, Agawam, MA) free of nematodes (Table 1). On the third day after emergence, and still in the absence of nematodes, seedlings were collected for root analysis.

On the third day after emergence, the seedlings not exposed to nematode infection were removed from soil and roots were washed several times with distilled cold water to remove soil debris. Then, roots were blotted in a paper towel to remove water, and then dipped in liquid nitrogen to quench all metabolic processes and prevent any stress response from sample processing. The frozen roots were then lyophilized for 48 hr, at −40 °C, and 0.05 MPa, to remove all water and permanently quench metabolism. Once the samples were dried, any remaining soil debris was removed from the roots using forceps. All roots were collected from each plant and their dry weight was recorded. Roots of six plants with similar size (fresh and dry weight) were selected to represent each of the nine genotypes and each of these six root samples was used separately for metabolite extraction and analysis.

The water content of the roots was calculated using the dry mass and used in determination of the methanol, chloroform, and water ratio (2:2:1.8). Methanol–chloroform–water extractions of the dried rootstocks were performed as described by Kim et al. (2010). Between 3 and 5 mg of dried roots were grinded using mortar and pestle. The exact masses of each replicate were recorded. The polar solvents were added in the mortar in order to recover as much plant materials as possible. The solution was then transferred to a glass conical vial containing chloroform and water. After vortex-mixing, the samples were kept on ice for 10 min. The samples were centrifuged at 4 °C at 2,000×g for 10 min. The polar and nonpolar phases of the sample were separated, and the polar solvents of each sample were removed using a Vacufuge Plus concentrator (Eppendorf AG, Hamburg, Germany).

Sample preparation and NMR spectroscopy followed the procedure described by Mahmud et al. (2015). After the solvents were completely removed from the polar fraction, 620 μL of NMR buffer (100 mM sodium phosphate buffer (pH 7.3), 1 mM 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid (internal standard, TMSP; CAS Registry No. 24493-21-8), and 0.1% sodium azide, in 99.9 atom % D₂O) were added to each sample. The samples were then vortex-mixed and centrifuged to remove any remaining undissolved material. From each sample, 600 μL were transferred into 5 mm NMR tubes (Norell). The NMR spectra were recorded on a Bruker Avance III spectrometer (Bruker Corporation, Billerca, MA, USA) operating at 700 MHz. The first increment of a presat-noesy experiment was acquired using the standard Bruker noesypr1d pulse sequence. This resulted in a one-dimensional ¹H spectrum (metabolic profile) for each sample (9 genotypes × 6 replicates). All data were collected using a spectral width of 16.0 ppm and 64K points resulting in an acquisition time of 2.9 sec. The spectra were collected with 120 scans, 4 dummy scans, 3 sec relaxation delay, and pre-saturation at the residual water frequency. The 90° pulse widths were measured for each sample using the automatic pulse calculation experiment (pulsecal) in TopSpin 2.1.1 (Bruker Bio Spin, Billerica, MA, USA). Two-dimensional ¹H–¹³C heteronuclear single quantum correlation (HSQC) data were collected for a representative CA sample and one for each cultivar using the Bruker hsqcedetgpsisp2.2 pulse sequence. The ¹H was observed in the F2 channel with a spectral width of 11 ppm, whereas the ¹³C was observed in the F1 channel with a spectral width of 180 ppm.

After the ¹H NMR data was collected on each of the 54 individual samples in this study, the first step was to generate bucket tables using AMIX software (version 3.9.7, BrukerBioSpin). Once data were normalized to total intensities, spectra were binned into 0.01 ppm wide buckets over the region of 0.5–10.0 ppm using advanced bucketing in AMIX. Two exclusions were made, one for the water region (4.75–4.90 ppm) and one for methanol contamination (3.39–3.27 ppm). Next, principal component analysis (PCA) was performed on the bucket tables generated from AMIX using MetaboAnalyst3.0 (Xia and Wishart, 2016). The Mahalanobis distance (D _M), two-sample Hotelling’s T2 statistic (T2), F values (Ft) and critical F values (Fc) were calculated using MatLabR2010b.31 for each score plot generated during the analysis (Goodpaster and Kennedy, 2011).

The next step was to identify the metabolites that were significantly different in concentration when the resistant CA and susceptible CL lines were compared. MetaboAnalyst3.0 was used to determine whether or not the changes in bucket intensities were statistically significant. Buckets with corresponding p values < 0.05 were considered statistically significant. Lastly, the metabolites corresponding to the peaks in the buckets were identified using the Biological Magnetic Resonance Data Bank (BMRB) (www.bmrb.wisc.edu/metabolomics/), Madison Metabolomics Consortium Database (MMCD) (http://mmcd.nmrfam.wisc.edu/), ChenomX NMR Suite (ChenomX Inc., Edmonton, AB, Canada), and published studies (Mahmud et al., 2014). Metabolic data interpretation was facilitated with the Metabolic Pathway Analysis (MetPA) component of MetaboAnalyst3.0 (Xia and Wishart, 2016).

Amino acids have been known to influence the development of nematodes (Sharma, 1998). Some amino acids (such as glutamine) enhance the development of nematodes like Heterodera schachtii while others (including methionine, phenylalanine, lysine, and tryptophan) inhibit the development of nematodes (Sharma, 1998; Betka et al., 1991). In this study, different amino acid metabolites such asparagine, arginine, valine, glutamine, isoleucine, alanine, and lysine were identified in higher concentrations in CA PIs when compared with both “Charleston Gray” and “Crimson Sweet”. The amino acids asparagine and arginine were identified in higher concentrations only in CA PI 189225 (1832).

Asparagine concentrations were 21 times higher in PI 189225 (1832) when compared with Charleston Gray and 47 times higher when compared with Crimson Sweet. Statistically significant metabolites were analyzed using MetaboAnalyst3.0 and KEGG Pathway database to identify the significant metabolites’ pathways. A KEGG Pathway database search showed that asparagine plays an important role in alanine, aspartate, and glutamate metabolism, all of which are involved in the amino acid metabolism. Aminoacyl-tRNA biosynthesis is another important metabolic pathway and in which asparagine plays an important role in the translation process (Fig. 2).

A closer look at the biochemical pathways associated with asparagine shows that it acts as a precursor for aspartate which is further used in the biosynthesis of arginine (Berg et al., 2002). Arginine is also found in much higher concentration in the same PI 189225 (1832) and it was reported to have a nematicidal effect in a study performed by Sayed and Thomason (1988). That study showed the effectiveness of ascorbic acid, thiamine, arginine, and glutamic acid on egg hatch, development, and reproduction of Meloidogyne incognita. Foliar sprays were applied in different concentrations, with their results showing that ascorbic acid, arginine, and glutamic acid suppressed the number of root galls, females, and egg masses. The same study also showed that the highest effect of foliar sprays using ascorbic acid and arginine was observed when the treatment is applied before inoculation with the nematodes. Guo et al. (2013) identified two enzymes (arginosuccinase and two arginosuccinate synthase) that are involved in conversion of citrulline (nonessential amino acid produced from glutamine) into arginine.

Glutamine is another amino acid present in significantly higher concentrations in five PIs in the pair-wise analysis with Charleston Gray and Crimson Sweet. Although it is well known that glutamine can accumulate due to stressful conditions (Hildebrandt et al., 2015), few reports have previously mentioned increased levels of glutamine in different plant tissues as a result of nematodes infection. These studies could not find any nematicidal activity of glutamine. More specifically, sugar beets infected with nematodes showed a significant increase of glutamine, aspartic acid and glutamic acid (Roy, 1981). Similarly, another study performed by Doney et al. (1970) on sugar beets reported an increased concentration in total amino acids (especially in the concentrations of glutamic acid, glutamine, and aspartic acid) in the fibrous roots of infected plants with nematodes.

Another amino acid, valine, was found in higher concentration in three CA PIs when compared with Charleston Gray, and in four of the PIs when compared with Crimson Sweet. KEGG metabolite pathway analysis revealed that valine was involved in the following pathways: pantothenate and CoA biosynthesis; valine, leucine, and isoleucine biosynthesis; valine, leucine and isoleucine degradation and; glucosinolate biosynthesis and aminoacyl-tRNA biosynthesis. In the literature, valine was reported to have a significant effect on reducing the number of galls, egg masses, and juvenile nematodes in DL-valine treated tomato plants (Hoque et al., 2014).

Even though there are no reports on the nematicidal activity of different amino acids in watermelon related literature, scientists have already studied the effect of different amino acids on the root-knot nematodes of other plants (tomato) (Hoque et al., 2014). Their results showed that some amino acids, especially dl-phenylalanine, l-proline, and l-histidine, promote plant growth and reduce galling incidence. Leonetti et al. (2010) showed that nitric oxide (NO) and hydrogen peroxide play an important role in tomato plants’ defenses against the root-knot nematodes. Nitric oxide is produced by three isoforms of NO synthase and all three isoforms use l-arginine and molecular oxygen as substrates (Forstermann and Sessa, 2012).

Fig. 2

Once plants are infected with M. incognita, more than 50% reduction of free sugars was reported when compared with healthy plants (Roy, 1981). Sugars, especially sucrose, represent the major source of carbohydrate input into nematode-induced feeding sites in Arabidopsis thaliana roots and metabolite analysis revealed increased levels of sucrose in syncytia and giant cells (Cabello et al., 2014). Carbohydrates were reported not to have a nematicidal effect; however, fructose, which does not have any nematicidal effect alone, makes DMDP (2R, 5R-dihydroxymethyl-3R,4R-dihydroxypyrrolidine) in combination with rotenone, a highly effective nematode control agent. DMDP can be applied in foliar application and translocated to the plant roots (Paranjape et al., 2014). In this study, fructose was found in higher concentrations in four amarus PIs when they were compared with Charleston Gray and in five PIs when they were compared with Crimson Sweet by using metabolite fold change analysis. Using the same metabolite fold change analysis, glucose was identified in higher concentrations in two CA PIs (PI 482259 and PI 482303) when they were compared with Charleston Gray and in six PIs when compared with Crimson Sweet. By contrast, glucose was present in Charleston Gray in higher quantities when compared with the three CA PIs (PI 189225, PI 482319, and PI 244018). Similarly, sucrose and raffinose were the two other sugars identified as significantly different metabolites when the CA PIs were compared with Charleston Gray and Crimson Sweet (Table 5).

Several organic compounds had higher concentrations in the amarus PIs when compared with Charleston Gray, namely: ethanolamine, succinate, 4-aminobutyrate, and formate (Table 5). More specifically, choline was found in significantly higher concentrations in three CA PIs and the others only in one PI. When the data comparison looked into the significantly different metabolites in the CA PIs when compared with Crimson Sweet, several other organic compounds were also identified in addition to the ones named previously: Although betaine, tartaric acid, malonate, ethanolamine, 2-oxoglutarate, and 4-aminobutyrate were found higher in the PI 1849 when compared with Crimson Sweet, there were not significant when compared with Charleston Gray (Table 5). Although we found conflicting results when compared the concentrations of choline, tartaric acid, and betaine in the PI’s versus Charleston Gray, choline was the most significant changing metabolite in three out of seven CA PIs when compared with the cultivars. The nematicidal effect of choline was previously reported in a perennial shrub found in the central Sahara region, named arghel (Solenostemma argel), which also contains acylated phenolic glycosides, flavonoids, monoterpene and triterpenoid saponin. Extracts from aerial parts (leaves) of argel were reported to be very effective (80–94% mortality rates after 72 hr exposure) against second stage juveniles of M. incognita (Abd El-Aziz, Ezz El-Din, 2007).

Tartaric acid was identified as a significantly different compound in one CA PI in the pair-wise analysis with Crimson Sweet. Previously, the nematicidal effect of tartaric acid was also reported by Jang et al. (2016). An exposure of 50 mmol/L of oxalic acid solution caused 96% mortality of M. incognita J2s at 1 day after treatment, whereas applications of 10 mmol/L resulted in mortality rates of 59.5%.

Lastly, another organic compound that was identified to be a significantly different metabolite when CA PIs were compared with Crimson Sweet was betaine. The use of seaweed extract on Meloidogyne javanica resulted in significant reduction in egg recovery. The effects of the seaweed extract were similar when betaine components of the seaweed extracts were used (Wu et al., 1998).

In summary, this study identified several metabolites in significantly higher concentrations in CA which were previously described in the literature to have nematicidal activity. Fold change analysis showed significant differences among some other compounds that could not be identified using Chenomx or available online databases. This study elucidated the richness of metabolites in roots of CA watermelons collected in the wild in southern Africa versus the limited number of metabolites in two major watermelon cultivars. The metabolite diversity between the CA PIs may be the result of the high genetic diversity that exists within the CA genome. The difference between the CA and the cultivars may have resulted from the loss of multiple genes during the domestication process of the watermelon. Despite their wide genetic diversity, CA and watermelon cultivars are readily crossable with each other. However, the higher amounts of metabolites in CA that have been previously described in the literature to have nematocidal activity could not be directly associated with any effect on nematodes. The CA PIs have proved useful as rootstocks for grafted watermelon in fields infested with RKN (Thies et al. 2012, 2015a, 2015b, 2015c), and are considered a useful germplasm for enhancing resistance to RKN and other soil-borne diseases in watermelon (Thies and Levi, 2003, 2007). Thus, further studies are underway to determine if any of the statistically significant metabolites that were present in CA PIs have nematicidal effects that might be related to the CA PIs’ resistance to RKN.