Effect of Carbon Dioxide Levels on Virulence and Physiological Parameters in Association with Stem Rot Disease of Groundnut

Groundnut (Arachis hypogaea L.), also known as the “king of oilseeds,” is an annual leguminous crop. It is extensively grown and distributed across more than eighty countries, primarily in tropical and subtropical regions (Madhusudhana, 2013). India ranks as the second-largest producer of groundnut globally, following China. The country plays a significant role in global oilseed production, with a total output of 25.5 million tons from 32.26 million hectares of land (Reddy and Maiti, 2023). In Telangana, groundnut occupies 2.61% of the total cultivated area and 28.18% of the state’s total oilseed acreage. The districts of Mahaboobnagar, Warangaland Nalgonda together account for 86.66% of the groundnut cultivation in the state (Jyothirmai et al., 2019).
       
The groundnut crop is affected by various diseases caused by fungi, bacteria, nematodes and viruses. These diseases adversely influence both the yield of groundnut pods and the quality of the resulting fodder (Vamshi et al., 2024a). Soil-borne diseases are major constraints in groundnut production. S. rolfsii is a soil-borne plant pathogen inciting root rot, stem rot, collar rot, wilt and foot rot diseases in over 500 plant species of agricultural and horticultural crops worldwide (Vamshi et al., 2024b). Stem rot also known as Sclerotium blight, Sclerotium rot, southern blight, southern stem rot, Sclerotium wilt, root rot, pod rot and white mold caused by Sclerotium rolfsii Sacc (Babu and Deepika, 2022). The development and survival of S. rolfsii are favored by high temperatures and moisture. The disease typically manifests in crops around one month of age, where the fungal mycelium covers the stem near the soil surface. This mycelium releases organic acids that damage plant cells and cause necrosis. The key symptoms of the disease include the presence of mycelium and sclerotia on the affected tissues (Kumar et al., 2008). According to Akgul et al., (2011), stem rot results in crop losses of 27% in India, 10-50% in South America and over 25% in Australia, with losses in heavily infected fields potentially reaching as high as 80% (Yan et al., 2019; Mayee  and Datar, 1988). The disease is most prevalent during the kharif season, often occurring alone or in conjunction with other diseases and it can lead to substantial yield reductions. Stem rot is considered one of the most destructive and widespread diseases affecting groundnut crops, especially in the northwestern plain region of Rajasthan (Sharma et al., 2012).
       
Air is a crucial resource for the survival and growth of living organisms. The composition of trace elements in the air can fluctuate due to emissions from various industrial and urban activities (Chen et al., 2017). In developing nations, fossil fuels remain the primary energy source for industrial operations, leading to the release of significant amounts of CO₂. This has contributed to an increase of approximately 28% in the atmospheric CO₂ concentration, which has risen from over 400 ppm (parts per million) today, compared to 311 ppm in the mid-nineteenth century (NASA GISS). Globally climate change is expected to pull down groundnut productivity by 11-25% (Sudhalakshmi et al., 2022). If the current pattern of fossil fuel consumption and population growth persists, CO₂ levels in the atmosphere could reach 500-1000 ppm by the year 2100 (IPCC 2013). Such elevated CO₂ concentrations could affect plant growth and the interactions between plants and pathogens (Khan and Khan 2000). Although carbon dioxide is not toxic to plants, it can stimulate plant growth and increase biomass production (Dader et al., 2016). This growth stimulation occurs due to a higher rate of CO₂ fixation, which reduces photorespiration (Li et al., 2007). The enhanced rate of photosynthesis under higher CO₂ levels typically results in increased plant growth and biomass. The extent of this growth promotion varies depending on the plant type: C3 plants (which make up the majority of plant species) experience a 30–34% increase in growth, while C4 plants (such as grasses in the Poaceae family) show a 10-15% increase (Kimball, 1983; Prior et al., 2003). Additionally, elevated CO₂ can affect other physiological and biochemical processes in plants (Agarwal  and Deepak, 2003; Jiang et al., 2012). For example, higher CO₂ concentrations negatively impact stomatal conductance and transpiration rates (Ainsworth  and Rogers, 2007). Furthermore, increased CO₂ levels (500-600 ppm) promote the production of chlorophyll and other leaf pigments (Sun et al., 2015).
       
In this regard, the current study seeks to assess the virulence of Sclerotium rolfsii and the physiological responses of plants when exposed to elevated CO₂ concentrations of 400 ppm, 550 ppm and 700 ppm.

Treatment setup in open top chambers (OTC)
 
A pot culture experiment was conducted at ICRISAT during the rabi season of 2022-23 in open top chambers (OTC) to evaluate the virulence and physiological responses of groundnut to climate change and pathogen interactions. The study was performed at three different carbon dioxide concentrations (400 ppm, 550 ppm and 700 ppm) using the susceptible cultivar TMV 2 and the moderately resistant cultivar ICGV-14082. Seedlings that were 30-35 days old were inoculated by applying the pathogen inoculum to the subsurface of the soil. Observations were made on the incubation period (IP), days to permanent wilting (DPW), disease incidence, disease severity and mortality.
       
Disease severity assessments were made by using a 1-5 disease severity scale (Shokes et al., 1996). Where,
 
Severity scale       Description
 
1                              A healthy plant
2                              Lesions on stems only
3                              Up to 25% of the plant symptomatic (wilted, dying or dead)
4                              26-50% of the plant symptomatic
5                              >50% of the plant symptomatic
The per cent disease severity was calculated using the formula (Le et al., 2012).
 

             

 
Where,
a = Number of diseased plants having the same degree of  infection.
b = Degree of infection.
A = Total number of plants examined.
K = Highest degree of infection.
 
Estimation of physiological changes occurring in groundnut in response to climate change and pathogen interaction
 
Stomatal density and guard cell length
 
Suitable leaves were collected from groundnut seedlings at 0, 2, 4, 6, 8 and 10 days post-inoculation, grown in OTC chambers. The leaves were gently washed with running water to remove dust and debris, then allowed to air dry. A replica fluid, such as Quick Fix, was applied in a thin, even layer by placing a drop or two on the leaf surface and allowing it to dry completely. Once dry, the replica was carefully peeled off using forceps or fingers and placed onto a slide, then covered with a cover slip. The number of stomata in the microscopic field was counted to calculate the stomatal density, which was expressed as stomata per mm². Stomatal density and guard cell length were measured using Image-J software in the leaves of groundnut plants inoculated with the virulent S. rolfsii isolate (SrPWp) in OTC chambers at 2-day intervals.
 
Chlorophyll content index (CCI)
 
A portable, non-invasive and lightweight device (SPAD-502 chlorophyll meter, Konica-Minolta, Japan) was used to measure the Chlorophyll Content Index (CCI). Fully mature leaves were selected at 0, 2, 4, 6, 8 and 10 days post-inoculation from groundnut seedlings grown in OTC chambers. Three CCI readings were taken from each pot and the average value was calculated to determine the mean CCI for each pot.
 
Statistical analysis
 
Per cent data was converted into arc sin values and square root transformed values. Fischer’s method of analysis of variance was used for analysis and interpretation of the data (Gomez and Gomez, 1984). Other statistical analysis viz., OP STAT online statistical analysis program developed by Hissar Agricultural University, IBM SPSS and MS-excel were used to analyze the data.

The potential impact of climate change on plant-pathogen interactions and its effect on crop production is a topic of considerable debate. It presents a significant challenge for future programs aimed at managing diseases in the context of global environmental changes.
       
The results (Table 1) showed that the incubation period of the pathogen lengthened as CO₂ concentrations increased from 400 to 700 ppm in both the susceptible cultivar TMV-2 and the moderately resistant cultivar ICGV-14082. However, no change was observed in the days to permanent wilting in the moderately resistant cultivar, which remained greater than 32 across all three CO₂ levels. In contrast, in the susceptible cultivar, the days to permanent wilting increased from 6.42 at 400 ppm to 13.90 at 550 ppm and further to 16.58 at 700 ppm.


       
In the TMV-2 cultivar, exposure to 700 ppm of CO₂ resulted in the highest mean incubation period (IP) of 8.25 (Table 1) (Fig 1), the longest days to permanent wilting (DPW) of 16.58 (Table 1)and the lowest disease incidence (90.22) (Table 2), disease severity (88.36) (Table 3) and mortality (82.82) (Table 4) (Fig 1). These values were similar to those observed at 550 ppm CO₂, which showed a mean IP of 6.92, DPW of 13.90, disease incidence of 94.82, disease severity of 91.00and mortality of 86.17. In contrast, at 400 ppm CO₂, the lowest mean IP (5.33), DPW (6.42)and the highest disease incidence (100.00), disease severity (100.00) and mortality (100.00) were recorded. These findings suggest that the virulence of S. rolfsii decreased as CO₂ levels increased in the TMV-2 cultivar.








       
In the ICGV-14082 cultivar, exposure to 700 ppm of CO₂ resulted in the highest mean incubation period (IP) of 16.90 (Table 1), the longest days to permanent wilting (DPW) (>32) (Table 1) and the lowest disease incidence (42.52) (Table 2), disease severity (40.18) (Table 3) and mortality (14.50) (Table 4) (Fig 1). These values were similar to those observed at 550 ppm CO₂, which showed a mean IP of 13.80, DPW (>32), disease incidence of 46.77, disease severity of 46.01and mortality of 18.35. In contrast, at 400 ppm CO₂, the lowest mean IP (11.25) and the highest disease incidence (50.37), disease severity (49.79) and mortality (21.20) were recorded, while the DPW remained >32. These results suggest that the virulence of S. rolfsii decreased as CO₂ levels increased in the ICGV-14082 cultivar.
       
The disease incidence was reduced by 49.63% in the moderately resistant cultivar ICGV-14082 compared to the susceptible cultivar TMV-2 at 400 ppm CO₂. A similar pattern was observed at 550 ppm (48.05%) and 700 ppm (47.7%).
       
Disease severity decreased by 50.21% in ICGV-14082 relative to TMV-2 at 400 ppm CO₂, with a similar trend at 550 ppm (44.99%) and 700 ppm (48.18%).
       
Mortality was lower by 78.8% in ICGV-14082 compared to TMV-2 at 400 ppm CO₂ and this trend continued at 550 ppm (67.82%) and 700 ppm (68.32%).
       
Our results align with those of Griffin and Nair (1968), who found that the growth rate of Sclerotium rolfsii mycelium remained constant within oxygen concentrations ranging from 3% to 21%, but decreased progressively as the carbon dioxide concentration rose above approximately 0.03%. Similarly, Kritzman and Henis (1977) reported that the growth rate and sclerotial production of S. rolfsii were significantly inhibited at a CO₂ concentration of 10%. In the same vein, Imolehin and Grogan (1980) observed that there were no significant differences in radial growth and sclerotial production when oxygen concentrations ranged from 4% to 21% (normal air) and CO₂ levels were at 0.03%. However, when the oxygen concentration dropped below 4% and CO₂ exceeded 8%, both radial growth and sclerotial production were greatly reduced. Furthermore, Punja and Jenkin (1984) reported that sclerotial germination was completely inhibited at temperatures between 9-12°C, at  water potentials lower than -60 bars and under conditions of CO₂ and O₂ levels exceeding 20% and dropping below 3%, respectively.
 
Physiological changes
 
Chlorophyll content index (CCI)
 
Chlorophyll content index (CCI) was measured using a SPAD meter in the leaves of groundnut plants inoculated with the virulent S. rolfsii isolate (SrPWp) in the OTC chamber at two-day intervals (Fig 2).


       
In the TMV-2 cultivar, the highest average CCI of 33.23 µmol m² was recorded at 700 ppm of carbon dioxide, followed by 33.08 µmol m² at 550 ppm and 30.76 µmol m² at 400 ppm.
       
In the ICGV-14082 cultivar, the highest average CCI of 40.91 µmol m² was recorded at 700 ppm, followed by 39.96 µmol m² at 550 ppm and 34.41 µmol m² at 400 ppm.
       
The CCI was 11.86% higher in the infected ICGV-14082 cultivar compared to the infected TMV-2 cultivar at 400 ppm CO₂, with increases of 20.79% at 550 ppm and 23.11% at 700 ppm.
       
These results indicate that the Chlorophyll Content Index increased as carbon dioxide concentrations rise.
       
Our findings are consistent with those of Habash et al., (1995), who demonstrated that wheat grown under elevated CO₂ conditions exhibited higher levels of chlorophyll pigments. Similarly, De Costa  et al. (2003) reported that elevated CO₂ concentrations enhanced leaf chlorophyll content in rice. Furthermore, Haque et al., (2006) investigated the impact of elevated CO₂ (570 ± 50 mmol mol- ¹) on leaf chlorophyll (Chl), nitrogen (N) content and photosynthetic rate (PN) during the post-flowering phase of rice grown in an Open Top Chamber (OTC). They found that both Chl and N contents were highest at the time of flowering under elevated CO2 conditions, compared to ambient CO₂ levels (~365 mmol mol- ¹) in OTC and open field conditions.
 
Stomatal density
 
Stomatal density was measured using Image-J software in the leaves of groundnut plants inoculated with the virulent S. rolfsii isolate (SrPWp) in an OTC chamber at two-day intervals (Fig 3).


       
In the TMV-2 cultivar, the highest average stomatal density of 217.1 stomata mm^-2 was recorded at 400 ppm of CO₂, followed by 204.3 stomata mm^-2 at 550 ppm and 200.6 stomata mm^-2 at 700 ppm.
       
In the ICGV-14082 cultivar, the highest average stomatal density of 207.5 stomata mm^-2 was recorded at 400 ppm, followed by 194.0 stomata mm^-2 at 550 ppm and 189.9 stomata mm^-2 at 700 ppm.
       
The stomatal density was 4.62% lower in the infected ICGV-14082 cultivar compared to the infected TMV-2 cultivar at 400 ppm CO₂, with reductions of 5.30% at 550 ppm and 5.63% at 700 ppm. These results indicate that stomatal density decreased as CO₂ concentrations increased.
       
Our findings are consistent with those of Clifford et al., (1995), who observed that when groundnut plants were grown under irrigated conditions with unrestricted root systems, an increase in atmospheric CO₂ from 375 to 700 ppm reduced stomatal frequency on both leaf surfaces by up to 16%. Similarly, Gitz et al., (2017) reported that elevated CO₂ levels led to a reduction in both the initiation of stomatal development on the adaxial and abaxial surfaces, as well as a decrease in stomatal density and size.
 
Guard cell length
 
Guard cell length was measured using Image-J software in the leaves of groundnut plants inoculated with the virulent S. rolfsii isolate (SrPWp) in an OTC chamber at two-day intervals (Fig 4).


       
In the TMV-2 cultivar, the highest average guard cell length of 28.3 µm was recorded at 400 ppm of CO₂, followed by 20.9 µm at 550 ppm and 19.5 µm at 700 ppm.
       
In the ICGV-14082 cultivar, the highest average guard cell length of 26.4 µm was recorded at 400 ppm, followed by 19.1 µm at 550 ppm and 17.6 µm at 700 ppm.
       
The guard cell length was 7.19% shorter in the infected ICGV-14082 cultivar compared to the infected TMV-2 cultivar at 400 ppm CO₂, with reductions of 9.42% at 550 ppm and 10.79% at 700 ppm. These results suggest that guard cell length decreased as CO₂ levels increased.
       
Our findings are consistent with those of Gitz et al., (2017), who reported that elevated CO₂ led to a decrease in both the initiation of stomatal development on the adaxial and abaxial surfaces, as well as a reduction in stomatal density and size.

The virulence of Sclerotium rolfsii was reduced at higher carbon dioxide concentrations in both the TMV-2 and ICGV-14082 cultivars. As carbon dioxide levels increased, the chlorophyll content index in groundnut leaves also showed an increase. However, stomatal density and guard cell length in the leaves decreased with rising CO₂ levels. Since Sclerotium rolfsii is an oxygen-dependent pathogen, its virulence diminished as carbon dioxide concentrations increased.