Reduction of Carbon Assimilation and Water Use Efficiency in Groundnut Ringspot Virus (GRSV)-Infected Groundnut Varieties



Abstract Views
149

Downloads
70

Citations

Share

##plugins.themes.bootstrap3.article.sidebar##

Submitted Jun 6, 2024
Published Sep 18, 2024
10.24018/ejbio.2024.5.5.523

Abstract viewed = 149 times

Table of Contents

##plugins.themes.bootstrap3.article.main##

Groundnut (Arachis hypogaea L.) production is threatened by the advent of many emerging pests and diseases, key among them being Groundnut ringspot virus (GRSV). The numerous alternative host crops of the virus make its control particularly difficult, resulting in heightened virus transmission risk and increased yield loss. The mechanisms underlying yield loss, though not fully understood, have often been speculated to be a result of the reduction in photosynthesis and transpiration in infected leaves. This study was therefore aimed at testing the effect of GRSV infection on carbon assimilation and water use efficiency by four farmer preferred groundnut cultivars (Nkosuor, Otuhia, Sinka, and Yenyawoso) in Ghana. Seeds of virus indexed plants previously grown in a screen house were nursed in a screen house and artificially infected with GRSV by grafting infected groundnut scions onto them to be compared with plants onto which uninfected groundnut scions had been grafted. The rate of photosynthesis, transpiration, and stomatal conductance of the distal leaf of each plant was measured using the CO2/H2O gas analyzer (CID, Inc., Camas, WA, USA). The virus infection was found to reduce stomatal conductance, transpiration, net photosynthesis, and 100 seed weight in a genotype dependent manner. This highlights the effect of GRSV infection on water conservation through a reduction in transpirational water loss, constraining photosynthesis, or carbon assimilation in the process to confirm the trade-off between photosynthesis and transpiration through the stomata.

Keywords: Arachis hypogaea Carbon assimilation Groundnut ringspot virus Water use efficiency

Introduction

Plant disease burden has been on the rise with the advent of climate change (Jeger, 2022), often with devastating consequences on crop production and livelihood sustenance. Discovered in Ghana less than a decade ago, Groundnut ringspot virus, GRSV (Appiahet al., 2016), a Tospovirus, is one of the major emerging threats to groundnut production and its value chain in Ghana. Besides groundnut, GRSV has been reported to infect other economically important crops including tomato, sweet pepper, cocona, coriander, cucumber, and watermelon (Spadottiet al., 2014; Websteret al., 2015). These together with the numerous alternative weed host species limits effective control of the disease. GRSV infection is characterized by leaf distortion, chlorotic ringspots, necrosis, and severe stunting (Websteret al., 2015), resulting in acute yield losses in groundnut (Michelottoet al., 2019).

The mechanism(s) underlying the severe yield losses in GRSV-infected groundnut is/are not directly known, but leaf necrosis symptomatic of infection has been hypothesized to reduce reception of incident light radiation, transpiration, and photosynthesis (Michelottoet al., 2019), which ultimately constrains yield. Similarly, other symptoms, including chlorosis, leaf distortion, and ringspot, could potentially inhibit optimum leaf function. In grapevine, for example, iron (Fe) deficiency-induced chlorosis has been linked to reduced photosynthesis (Shahsavandiet al., 2020) through the degradation of photosynthetic apparatus (Bertaminiet al., 2001). In Areca catechu however, Fe deficiency linked chlorosis has been associated with reduced chlorophyll synthesis and degradation of the chloroplast (Liet al., 2021). Additionally, leaf xylem vessel size and leaf hydraulic conductance in Prunus persica (peach) have been found to be reduced by Fe chlorosis (Eichertet al., 2010). This also affects the water relations of peach, inducing a decline in transpiration, net photosynthesis, and water use efficiency (Eichertet al., 2010).

The effect of GRSV infection on transpiration in infected groundnut remains unknown. However, evidence from Murrayet al. (2016) on other virus pathosystems, such as susceptible Nicotiana tabacum and Arabidopsis thaliana (Col-0) infected with Tobacco mosaic virus (TMV) and Turnip vein clearing virus (TVCV), respectively, indicate a severe reduction in transpiration. This was a result of a reduction in stomatal density and stomatal index, which have earlier been reported to affect the water use efficiency (WUE) of the crop (Frankset al., 2015; Doheny-Adamset al., 2012) with a direct consequence on stomatal conductance.

According to Guerrieriet al. (2019), Water use efficiency (WUE) at the core of ecosystem functioning is reflected by the trade-off between photosynthesis and transpiration through foliage stomata. This highlights the importance of stomatal conductance to photosynthesis of crops. Yet, the question of the impact of GRSV infection on the cascade of events that lead to the carbon assimilation has remained outstanding for several years. This study, therefore, investigated the effect of GRSV infection on carbon assimilation and water use efficiency of graft-inoculated groundnut varieties.

Materials and Methods

Groundnut Varieties and Nursery Establishment

Seeds of four screenhouse-grown groundnut cultivars viz. Otuhia, Nkosuor, Sinka, and Yenyawoso were nursed three per pot, with a total of 40 pots for each cultivar. The pots were labelled and randomly arranged in the screenhouse at the Biotechnology and Nuclear Agriculture Research Institute (BNARI) of the Ghana Atomic Energy Commission. The pots were hand-watered every other day using a watering can, to ensure adequate moisture and monitored for germination. Three weeks after germination, 20 pots of each cultivar were selected, and the seedlings thinned out, leaving one seedling per pot.

Graft Inoculation

Each cultivar was divided into two sets of 10 pots. For one set, a healthy disease-free scion was side grafted onto the main stem of the seedling in each pot, this was repeated with PCR confirmed GRSV-infected scion for the other set. The pots were watered, after which each grafted plant was covered for one week with a transparent polybag to create a microclimate with high humidity.

Symptom Observation and PCR Detection

Symptoms were observed four weeks after inoculation, and the GRSV-infection status of the plants was confirmed with PCR using primers targeting the capsid protein gene (Boariet al., 2002). The primer pair used for the detection were NGRSVF (5′-GATCTAAGGATCCACCATGTCTAAGGTCAAGCT C-3′) and NGRSVR (5′-CCCTGCAGAGCTCTCATGC AACACCAGCAATCTTG-3′). PCR conditions used were as described by Boariet al. (2002).

Plant Measurements

Four weeks after the removal of the polybags, the rate of photosynthesis, transpiration, and stomatal conductance of the distal leaf of each plant was measured using the CO2/H2O gas analyzer (CID, Inc., Camas, WA, USA).

Yield Estimation

Yield was estimated by harvesting pods from each variety from both diseased and healthy plants. The seeds were then air-dried and shelled, and the average seed and the 100-seed weight for diseased and healthy plants were measured for each cultivar.

Data Analysis

The study involved the use of a two-factor experiment that was laid in a completely randomized design (CRD). Experimental units comprised individual groundnut plants. Each experimental unit constituted a replicate, and all treatments were replicated 10 times unless otherwise stated. Data residuals were tested for normality using histogram plots followed by Shapiro–Wilk’s test. The data were analysed using the analysis of variance of the respective experimental designs to determine the P values using R version 3.4.4 and RStudio version 1.1.447. Where the P value showed significant difference at 5% significance level, a mean separation was done using Tukey HSD.

To determine the effect of GRSV infection on the relationships between internal CO2 concentration and stomatal conductance, transpiration, or photosynthesis, the LM function of the R package stats (version 4.0.2) in R version 3.4.4 was used with RStudio version 1.1.447 to run linear regression analysis on individual predictors and for graphical representation of data.

Results

GRSV-infected plants of the four groundnut varieties expressed symptoms including irregular chlorotic patterns, stunting, and deformation of leaflets, indicative of Tospovirus infection (Fig. 1).

Fig. 1. Plants of Yenyawoso: a) Healthy plant b) GRSV-infected plant showing stunting, leaf deformation, and chlorosis.

Effect of Virus Infection on Stomatal Conduction

Stomatal conductance varied significantly (P ≤ 0.05) from a low value of 4 µmol/mol in Nkosuor to a high value of 163 milimol/m2/s in Yenyawoso. Generally, the non-infected plants had higher stomatal conductance than the infected plants, although the difference was statistically insignificant (P ≤ 0.05). Similarly, the interactive effect of variety and infection status of the plants were not statistically significant, but perceptible differences were recorded in all the treatments tested. Whilst infected Yenyawoso plants recorded 109 milimol/m2/s stomatal conductance, the non-infected recorded 217 milimol/m2/s stomatal conductance (Fig. 2).

Fig. 2. Effect of Groundnut ringspot virus (GRSV) infection on stomatal conductance of different varieties groundnut cultivars.

In infected plants of Otuhia, stomatal conductance was 10 µmol/mol, whilst in non-infected, it was 70 µmol/mol. Infected Nkosuor was 5 milimol/m2/s, with non-infected being 3 milimol/m2/s, likewise infected Sinka was 24 milimol/m2/s, but the non-infected was 20 milimol/m2/s (Fig. 1).

Effect of GRSV Infection on Internal CO2 Concentration

The effect of GRSV infection on the internal CO2 concentration (IntCO2) in the leaves of the different groundnut cultivars was not significantly different at P ≤ 0.05 despite some observed differences. The IntCO2 ranged from a low of 2213 µmol/mol in non-infected Yenyawoso to a high of 4255 µmol/mol in infected Nkosuor. Also, the interactive effect of cultivar and GRSV infection on IntCO2 was not significantly different. In Nkosour and Otuhia, the IntCO2 in both the infected and non-infected were similar at ~4250 µmol/mol and ~3250 µmol/mol, respectively (Fig. 2), while in Yenyanwoso GRSV infection resulted in higher IntCO2 than in the non-infected crop, the opposite of this phenomenon was however observed in Sinka (Fig. 3).

Fig. 3. Effect of Ground nut ringspot virus (GRSV) infection on internal CO2 concentration in leaves of different groundnut cultivars.

Effect of GRSV Infection on Transpiration

The effects of GRSV infection and cultivar effect, as well as their interactive effect on transpiration rate, were not statistically significant (P ≤ 0.05). At ~0.073 and 0.041 milimol/m2/s, transpiration rates in both infected and non-infected plants of Nkosuor as well as that of Otuhia, respectively, were nearly identical (Fig. 4). In Yenyawoso and Sinka, variations in transpiration rate were observed. Transpiration rates in infected and non-infected Sinka were 0.049 and 0.225, respectively, whilst those in Yenyawoso were 0.039 and 0.094 for infected and non-infected, respectively (Fig. 4).

Fig. 4. Effect of Ground nut ringspot virus (GRSV) infection on transpiration in the leaves of different ground nut cultivars.

Effect of GRSV Infection on Net Photosynthesis (Pn)

Generally, net photosynthesis (Pn) was subzero in all treatments with significant (P ≤ 0.05) differences in Pn response to varietal and GRSV infection. Infected plants had much lower photosynthetic rates than non-infected plants. Pn in Sinka was −0.302 but reduced to −0592 when infected with GRSV. Similarly, in Nkosuor, it reduced from −0.219 in non-infected to −0.258, Otuhia from −0.376 to −0.394, and Yenyawoso from −0.087 to −0.275 in infected plants, respectively (Fig. 5).

Fig. 5. Effect of Ground nut ringspot virus (GRSV) infection on net photosynthesis in the leaves of different ground nut cultivars.

Relationship Between Stomatal Conductance and Internal CO2 Concentration

There was an inverse relationship between stomatal conductance and IntCO2, with stomatal conductance generally declining linearly with increasing IntCO2 in both GRSV infected and non-infected crop. Whilst the stomatal conductance was higher in the non-infected plants than the infected at most IntCO2, the rate of decline of stomatal conductance with increasing IntCO2 was higher than in the infected plants (Fig. 6).

Fig. 6. Fitted linear regression showing the impact of Ground nut ringspot virus infection on the relationship between stomatal conductance and internal CO2 in groundnut leaves.

Relationship Between Transpiration and Internal CO2 Concentration

Similar to the stomatal conductance, the transpiration rate declined linearly with increasing IntCO2 concentration, with mean transpiration higher at most IntCO2 in the non-infected than in the infected plants (Fig. 6). The rate of decline in transpiration rate with increasing IntCO2 was steeper in the non-infected than in the infected (Fig. 7).

Fig. 7. Fitted linear regression showing the impact of Groundnut ringspot virus infection on the relationship between transpiration rate and internal CO2 in groundnut leaves.

Relationship Between Rate of Photosynthesis and Internal CO2 Concentration

The rate of photosynthesis also declined linearly with increasing IntCO2. At most IntCO2, Pn in the non-infect crop was higher than in the infected crop, but whilst the rate of decline in the infected crop was very marginal, in the non-infected crop, the rate was much steeper (Fig. 8).

Fig. 8. Fitted linear regression showing the effect of Ground nut ringspot virus infection on the relationship between the rate of photosynthesis and internal CO2 in groundnut leaves.

Effect of GRSV Infection on the Yield of the Groundnut Varieties

The GRSV-infected plants of the four cultivars had numerous pegs, a few single-seeded pods and several unfulfilled pods when compared with healthy plants (Fig. 9). The infection significantly (P < 0.001) reduced the 100-seed weights for all virus-infected plants of the four cultivars (Fig. 10). The least reduction in yield (18%) was associated with cultivar Otuhia whilst Nkosour showed the greatest (46%) reduction in average yield per plant.

Fig. 9. Harvested pods of Yenyawoso: a) Healthy plants b) plants of GRSV-infected plants showing numerous pegs, small-sized and numerous single-seeded pods.

Fig. 10. Reduction in 100-seed weight of GRSV-infected groundnut varieties.

Discussion

Gaseous (CO2, O2, and H2O vapour) exchange between sub-stomatal cavity and the extracellular environment is essential for many metabolic and catabolic processes in vascular plants. These gaseous fluxes in the leaf are regulated by the stomata (Murrayet al., 2016), with the net influx of CO2 or efflux of water vapour through the stomata measured as the stomatal conductance (Hernandezet al., 2016). Often, differences in stomatal conductance are underpinned by genotypic differences (Gilbertet al., 2011). In this study, significant variations in stomatal conductance were apparent among the different groundnut cultivars, highlighting genotypic influences, an observation similar to earlier reports in soybean by Gilbertet al. (2011). Stomatal conductance is a function of stomatal density, stomatal size, and stomatal aperture (Ziegler & Farquhar, 1987) determined largely by genotype. Little is known about the stomatal diversity of the groundnut cultivars used in this study. Thus, the reason underlying the differences in stomatal conductance is subject to speculation. This notwithstanding, GRSV infection reduced stomatal conductance by at least 50% in Otuhia and Yenyawoso, indicating the susceptibility of the two cultivars to GRSV infection. This agrees with earlier work by El Aou-ouadet al. (2017), which showed the inhibitory effect of virus infection on stomatal conductance.

With stomatal conductance and transpiration proceeding through the same stomatal route, a positive correlation between the two phenomenon is often expected. However, the pattern of transpiration of the groundnut cultivars did not directly mimic that in the stomatal conductance, with the exception of Nkosuor and Yenyawoso. Here, the transpiration rates in both infected and non-infected cultivars followed the same pattern as their stomatal conductance. Contrarily, in Otuhia, where stomatal conductance in the non-infected plants was over three-fold that of the infected plants, the transpiration rates did not differ with GRSV infection. Similarly, in Sinka, stomatal conductance varied slightly between the infected and non-infected plants but transpiration rate in the non-infected plants was about three and half-fold that of the infected. These results suggest a non-positive genotypic effect in the relationship between transpiration and stomatal conductance, contrary to observation in sugarcane by Endreset al. (2010). Likewise, stomatal conductance did not correlate positively with internal CO2 concentration. No clear pattern in internal CO2 was discernible among the infected and non-infected plants.

The observation of negative net photosynthesis in all treatments in this study, where photosynthetically active radiation (PAR) ranged from 20.5–147.4 millimol/m2 and atmospheric CO2 concentration was between 1773.2–2195.9 µmol/mol, suggests respiration exceeded photosynthesis at the prevailing conditions. This notwithstanding, GRSV infection led to much lower net photosynthesis compared to the non-infected plants in all cultivars. This corroborates the report of an earlier study in grapevines where viral infection resulted in nearly 50% reduction in photosynthesis (Sampolet al., 2003). The reduction in photosynthesis in the groundnut cultivars showed a positive association with the effect of GRSV infection on stomatal conductance.

Conclusion and Recommendations

The study showed that stomatal conductance in groundnut is genotype dependent with Yenyawoso being the cultivar with the highest stomatal conductance. GRSV infection effect on stomatal conductance was genotype dependent as Otuhia and Yenyawoso showed at least 50% reduction in stomatal conductance. Also, stomatal conductance did not correlate with transpiration and internal CO2 concentration. On the other hand, Net photosynthesis was reduced by the virus infection, indicating carbon assimilation reduction effect by the virus. Reduction in stomatal conductance resulted in decreases in net photosynthesis.

To be able to ascertain the factors underpinning the genotypic differences in stomatal conductance, the stomatal morphology and count of the different groundnut genotypes must be studied to facilitate the selection of genotypes for future genetic improvement programmes. Similarly, further studies are required to determine the PAR and atmospheric CO2 concentration required for net positive photosynthesis.

References

  1. Appiah, A. S., Offei, S. K., Tegg, R. S., & Wilson, C. R. (2016). Varietal response to groundnut rosette disease and the first report of Groundnut ringspot virus in Ghana. Plant Disease, 100, 946–952.
     Google Scholar
  2. Bertamini, M., Nedunchezhian, N., & Borghi, B. (2001). Effect of iron deficiency induced changes on photosynthetic pigments, ribulose- 1, 5-bisphosphate carboxylase, and photosystem activities in field grown grapevine (Vitis vinifera L. Cv. Pinot noir) leaves. Photosynthetica, 39(1), 59–65.
     Google Scholar
  3. Boari, A. J., Maciel-Zambolim, E., Lau, D. D., Lima, G. S. A., Kitajima, E. W., Brommonschenkel, S. H., & Zerbini, F. M. (2002). Detection and partial characterization of an isolate of Groundnut ringspot virus in Solanum sessilif lorum. Fitopatologia Brasileira, 27, 249–253.
     Google Scholar
  4. Doheny-Adams, T., Hunt, L., Franks, P. J., Beerling, D. J., & Gray, J. E. (2012). Genetic manipulation of stomatal density influences stomatal size, plant growth and tolerance to restricted water supply across a growth carbon dioxide gradient. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, 367, 547–555.
     Google Scholar
  5. Eichert, T., Peguero-Piña, J. J., Gil-Pelegrín, E., Heredia, A., & Fer- nández, V. (2010). Effects of iron chlorosis and iron resupply on leaf xylem architecture, water relations, gas exchange and stomatal performance of field-grown peach (Prunus persica). Physiologia Plantarum, 138, 48–59.
     Google Scholar
  6. El Aou-ouad, H., Pou, A., Tomás, M., Montero, R., Ribas-Carbo, M., Medrano, H., & Bot, J. (2017). Combined effect of virus infection and water stress on water flow and water economy in grapevines. Physiologia Plantarum, 160, 171–184.
     Google Scholar
  7. Endres, L., Silva, J. V., Ferreira, V. M., & De Souza Barbosa, G. V. (2010). Photosynthesis and water relations in Brazilian sugarcane. Open Agricultural, 4(1), 31–37.
     Google Scholar
  8. Franks, P. J., Doheny-Adams, T. W., Britton-Harper, Z. J., & Gray, J. E. (2015). Increasing water-use efficiency directly through genetic manipulation of stomatal density. New Phytologist, 207, 188–195.
     Google Scholar
  9. Gilbert, M. E., Zwieniecki, M. A., & Holbrook, N. M. (2011). Indepen- dent variation in photosynthetic capacity and stomatal conductance leads to differences in intrinsic water use efficiency in 11 soybean genotypes before and during mild drought. Journal of Experimental Botany, 62(8), 2875–2887.
     Google Scholar
  10. Guerrieri, R., Belmecheri, S., Ollinger, S. V., Asbjornsen, H., Jennings, K., Xiao, J., Stocker, B. D., Martin, M., Hollinger, D. Y., Bracho- Garrillo, R., Clark, K., Dore, S., Kolb, T., Munger, J. W., Novick, K., Richardson, A. D. (2019). Disentangling the role of photosynthesis and stomatal conductance on rising forest water-use efficiency. Proceedings of the National Academy of Sciences, 116(34), 16909–16914. https://doi.org/10.1073/pnas.1905912116.
     Google Scholar
  11. Hernandez, M. J., Montes, F., Ruiz, F., Lopez, G., & Pita, P. (2016). The effect of vapour pressure deficit on stomatal conductance, sap pH and leaf-specific hydraulic conductance in Eucalyptus globulus clones grown under two watering regimes. Annals of Botany, 117(6), 1063–1071.
     Google Scholar
  12. Jeger, M. J. (2022). The impact of climate change on disease in wild plant populations and communities. Plant Pathology, 71(1), 111–130.
     Google Scholar
  13. Li, J., Xianmei, C., Xiaocheng, J., Liyun, L., Haowei, C., Weiquan, Q., & Meng, L. (2021). Iron deficiency leads to chlorosis through impacting chlorophyll synthesis and nitrogen metabolism in Areca catechu L. Frontiers of Plant Science, 12, 1577.
     Google Scholar
  14. Michelotto, M. D., Carrega, W. C., Lamana, L. E. P., de Souza, T. M., de Godoy, I. J., Almeida dos Reis, L. N., Sales, A. L. M., & Carvalho, R. C. P. (2019). Losses caused by Groundnut ringspot tospovirus in peanut crop in the State of São Paulo. Semina: Ciências Agrárias, 40, 3429–3442.
     Google Scholar
  15. Murray, R., Emblow, M., Hetherington, A., & Foster, G. D. (2016). Plant virus infections control stomatal development. Scientific Reports, 6, 34507. https://doi.org/10.1038/srep34507.
     Google Scholar
  16. Sampol, B., Bota, J., Riera, D., Medrano, H., & Flexas, J. (2003). Analysis of the virus-induced inhibition of photosynthesis in malmsey grapevines. New Phytologist, 160, 403–412.
     Google Scholar
  17. Shahsavandi, F., Eshghi, S., Gharaghani, A., Ghasemi-Fasaei, R., & Jafarinia, M. (2020). Effects of bicarbonate induced iron chlorosis on photosynthesis apparatus in grapevine. Scientia Horticulturae, 270, 109427.
     Google Scholar
  18. Spadotti, D., Leão, E., Rocha, K., Agenor, P., & Krause-Sakate, R. (2014). First report of Groundnut ringspot virus in cucumber fruits in Brazil. New Disease Reports, 29, 25. https://doi.org/10.5197/j.2044-0588.2014.029.025.
     Google Scholar
  19. Webster, C. G., Frantz, G., Reitz, S. R., Funderburk, J. E., Mellinger, H. C., McAvoy, E., Turechek, W. W., Marshall, S. H., Tantiwanich, Y., McGrath, M. T., Daughtrey, M. L., Adkins, S. (2015). Emergence of Groundnut ringspot virus and Tomato chlorotic spot virus in Vegetables in Florida and the Southeastern United States. Phytopathology, 105(3), 388–398.
     Google Scholar
  20. Ziegler, G. D., Farquhar, I. R., Cowan and Eduardo, (1987). Stomatal Function, pp. 29, Stanford, California: Board of Trustees of the Leland Stanford Junior University. ISBN 9780804713474.
     Google Scholar