Geminivirus infection in Jatropha
The Jatropha plants of different accessions like CP9, PCM, Ranpur, Shyamalaji, and Zanjmer were infected by geminivirus at Nesvad, Chorvadla and Bhadraval field sites of CSIR-CSMCRI with >70% geminivirus disease incidence . The symptoms were similar in all accessions with severe leaf curling. In the present study, we used only CP9 accession from Nesvad location as it is economically more important due to its high seed and oil yield . The healthy plants showed no leaf curl mosaic symptoms (Fig. 1A). In the case of younger plants, all the leaves showed the prominent yellow mosaic curl symptoms (Fig. 1B), while in old plants, only the new young leaves showed the prominent yellow mosaic curl symptoms (Fig. 1C). The virus responsible for causing the leaf curl disease was identified as JLCuGV in our earlier study . The collected Jatropha leaf samples (three replicates each of healthy and infected leaf tissue) were checked for geminivirus infection. Only those samples showing leaf curl symptoms were identified as positive by PCR (Fig. 1D) and were used for the metabolite analysis.
Metabolite profiling in healthy and infected Jatropha leaf
The metabolite analysis of geminivirus infected and healthy Jatropha leaf tissue revealed an accumulation of 98 total metabolites. (Table 1). Out of them, 49 metabolites were detected in both tissues, 28 and 21 unique metabolites in healthy and infected tissue, respectively (Fig. 2A, Table 1). All the detected metabolites were categorised in eight different groups like alkanes, amino acids, carboxylic acids, fatty acids, fatty alcohols, polyols, polyphenols and sugars. The compounds like D-galactopyranoside and 2-ketoglutaric acid were detected in higher amount in healthy tissue only, while hexonic acid and 4- Aminobutyric acid were two compounds which were detected in higher concentration in only infected tissue (Table 1).
Sugars were the largest group of compounds, and the amount of sugar was more in the infected tissue (48%) than healthy tissue (35%; Fig. 2B). Twelve different sugars were identified in both healthy and infected tissue. In the infected tissue, sugars like fucopyranose, trehalose, glyceryl-glycoside, and mannobiose decreased, while sucrose, glucose, galactoside, glucopyranose, lactose and fructose increased. L-fucopyranose decreased by 2-fold, while lactose and fructose increased significantly by 2.14- and 3.23-fold, respectively (Table 2, Fig. 3).
The carboxylic acid was the second largest group identified with 26% and 16% concentration in the healthy and geminivirus infected tissue, respectively (Fig. 2B). Fourteen carboxylic acids were detected in both healthy and infected tissue. Carboxylic acids like glutaric acid, malic acid, quinic acid, pentadecafluorooctanoic acid, succinic acid, citric acid and threonic acid were significantly decreased, while glyceric acid was significantly increased in infected tissue (Table 2, Fig. 3). In the infected tissue maximum decrease of 24.5-fold was observed in glutaric acid, >2-fold decrease in malic acid, quinic acid, galactaric acid, methoxyacetic acid, pentadecafluorooctanoic acid, succinic acid as compared to healthy tissue. The glyceric acid, gluconic acid, fumaric acid, and shikimic acid increased by <1.2-fold in infected tissue (Table 2).
In addition to sugars and carboxylic acids, the next larger group was polyols. The polyols accumulation was more in healthy tissue (23%) as compared to infected tissue (19%; Fig. 2B). Total nine polyols were detected in both healthy and infected tissue. Amongst them, galactinol, triethylene and myo-inositol decreased significantly, while deoxypentitol increased significantly in the infected tissue (Fig.3). Galactinol decreased by 2.04-fold, while deoxypentitol increased by 2.77-fold in infected tissue (Table 2).
Similarly, the concentration of fatty acids was also lower in infected tissue (6%) as compared to healthy tissue (8%). Total five fatty acids were detected, and among them, docosanoic acid, palmitic acid, and stearic acid were significantly decreased by >1.5-fold, while butanoic acid octatriacontyl pentafluoropropionate were unaffected (Table 2, Fig. 3). The polyphenols were more in infected tissue (10%) than healthy tissue (6%). Total three polyphenols detected in both tissue and among them, quercetin was increased significantly by 3.7-fold in infected tissue (Table 2, Fig. 3).
The other compounds like fatty alcohols, alkanes and amino acids were detected in less quantity (< 2%) in both the healthy and infected tissue, furthermore their concentration decreased on the infection (Fig. 2B). Amongst them, L-serine is the most affected compound with a 19.67-fold decrease in infected tissue (Table 2). The identified compounds mostly participate in sugar metabolism and TCA cycle. Their variation in healthy and infected tissue is shown in the interlinked pathway of sugar metabolism and TCA cycle (Fig. 4). Sugars (glucose, fructose, sucrose, galactoside, glucopyranose and lactose) were mostly increased in infected tissue, while the compounds generated from the metabolism of these sugars like sugar alcohols (butanetriol, erythritol, myo-inositol, galactinol) and carboxylic acids (galactaric acid threonic acid and ribonic acid) were higher in healthy tissue. Glutaric acid, a by-product of amino acid metabolism, was also higher in healthy tissue. Similarly, compounds of TCA cycle (citric acid, succinic acid and malic acid) and derivatives of fatty acids (docosanoic acid, dihydroxybutanoic acid, palmitic acid and stearic acid) were more in healthy tissue (Fig. 4).