Cotton leafroll dwarf virus (CLRDV; family Solemoviridae, genus Polerovirus) is known to cause cotton blue disease (CBD), and its occurrence was first reported in Africa in 1949, followed by reports of the disease in Asia and South America (Fang et al., 2010). Cotton yield losses up to 80% have been observed from CLRDV in South America (Silva et al., 2008), and the virus is now an emerging threat to cotton production in the United States (Avelar et al., 2019). CLRDV was first reported in Alabama in 2017 (Avelar et al., 2019) and has subsequently been documented in 10 other cotton producing states, with disease incidence varying from less than 1% to more than 20% across the United States cotton belt (Aboughanem-Sabanadzovic et al., 2019; Tabassum et al., 2019; Alabi et al., 2020; Ali et al., 2020; Ali and Mokhtari, 2020; Faske et al., 2020; Iriarte et al., 2020; Price et al., 2020; Thiessen et al., 2020; Wang et al., 2020). Interestingly, cotton plants infected with the United States strains of CLRDV showed different symptoms from CBD; therefore, the disease caused by this virus has been named as cotton leafroll dwarf disease (CLRDD; Brown et al., 2019). Numerous symptoms have been associated with CLRDD. A few of the notable symptoms include reddening of leaves and petioles, leaf wilting, subsequent drooping, crinkling, and deformation of the leaves above the nodes, which had reddened leaves, development of intense green foliage, and shortening of upper internodes resulting in stunted plants (Tabassum et al., 2019, 2020; Bag et al., 2021). Severe disease infection could result in reduced boll set and lower seed-cotton yield per plant (Avelar et al., 2020). The disease symptomology can vary substantially depending upon plant developmental stage at the time of infection, stage of disease progression, location, and variety.

CLRDV is transmitted by the cotton aphid (Aphis gossypii L.; Avelar et al., 2020) in a circulative and persistent manner (Silva et al., 2008). CLRDV is a positive sense, single-stranded RNA virus, and it is a phloem-limited virus, meaning it replicates and circulates mainly in the phloem tissue of the host plant (Silva et al., 2008; Jiménez et al., 2021). In the United States, the partial genome sequence of CLRDV was first sequenced from isolates collected from Alabama (Avelar et al., 2020), and the complete genome was subsequently completed from an isolate collected in Georgia (Tabassum et al., 2020, 2021). The genomic sequence of the CLRDV isolate from Georgia was between 95 and 98% identical to the genome of other CLRDV isolates reported in the United States (Alabama, GenBank accession number MN071395; Texas, MN872302) and South America (KF359947, KF906261, KF906260, NC_014545, GU167940, and HQ827780; Tabassum et al., 2020, 2021). Despite obvious genomic similarity, there are three unique genotypes of CLRDV recognized globally: “atypical,” “typical,” and “CLRDV-US” (Avelar et al., 2020; Iriarte et al., 2020).

Since CLRDD is new to the United States, there is very little information available on the physiological response of cotton to the disease. With other viral plant diseases that visibly affect foliage, significant reductions in net photosynthesis (A_n) have been documented (Ryšlavá et al., 2003). Furthermore, virus-induced declines in A_n have been associated with reduced efficiency of the light dependent processes of the thylakoid reactions, lower stomatal or mesophyll conductance to CO₂, or reduced activity of the carbon fixation reactions of the Calvin cycle (Sampol et al., 2003; Moutinho-Pereira et al., 2012; Souza et al., 2017). Another important observation from the previously noted studies is that the degree of photosynthetic inhibition observed and the primary factor contributing to photosynthetic decline can vary substantially depending on plant species, cultivar, and virus. Because CLRDV causes visible changes in leaf coloration and turgor, it is reasonable to assume that CLRDV-induced photosynthetic decline might be associated with reductions in stomatal conductance (g_s) and possibly the activity of the thylakoid reactions.

During the 2019 and 2020 growing seasons, experiments containing multiple cotton cultivars were conducted at field sites in Tifton, GA. In both seasons, plants with CLRDD symptoms of varying severity were consistently observed in the early season for cotton cultivar DG 3615 B3XF (symptomatic cultivar, S), whereas other cultivars at the same sites exhibited 0% disease incidence (asymptomatic cultivars, A). Since some cotton cultivars have recently been shown to differ in their stomatal response to vapor pressure deficit (Shekoofa et al., 2021), otherwise normal plants of A and S cultivars may exhibit inherent differences in gas exchange responses under the same field conditions. It was hypothesized that diseased plants of the symptomatic cultivar would exhibit lower light-saturated photosynthetic rates, reduced photochemical efficiencies of photosystem II, declines in stomatal conductance, increased leaf temperature, and reduced productivity relative to plants of the same cultivar without conspicuous disease symptoms. We also hypothesized that asymptomatic cultivars would exhibit more conservative gas exchange responses (lower g_s) than otherwise normal plants of the symptomatic cultivar under field conditions. Thus, the objectives of the current study were to determine gas exchange, chlorophyll fluorescence, leaf temperature, and yield responses for a susceptible cultivar at multiple stages of disease progression and for otherwise healthy-appearing plants of symptomatic and asymptomatic cultivars.

In the current study, it was hypothesized that cotton plants of the susceptible cultivar showing CLRDD symptoms would exhibit lower light-saturated photosynthetic rates, reduced photochemical efficiencies of photosystem II, declines in stomatal conductance, increased T_leaf, and reduced productivity relative to plants of the same cultivar without conspicuous disease symptoms. The data presented herein support our hypothesis. For example, net assimilation (A_n), stomatal conductance (g_s), and primary photochemistry (Φ_PSII and ETR) decreased significantly as CLRDD symptoms progressed to more advanced stages. In fact, A_n was essentially eliminated in diseased plants for stages S₂ through S₄ on the initial sample date in both years of our experiment (Figures 2, 3). Significant reductions in A_n, g_s, Φ_PSII, and ETR have been reported in viral diseases such as the sugarcane yellow leaf virus (family Solemoviridae, genus Polerovirus) in sugarcane (Lehrer and Komor, 2008), grapevine leaf roll-associated viruses (family Closteroviridae, genus Ampelovirus), grapevine fan leaf virus (family Comoviridae, genus Nepovirus), and grapevine fleck virus (family Tymoviridae, genus Maculavirus) in grapevine (Sampol et al., 2003; Bertamini et al., 2004), pepper mild mottle virus and paprika mild mottle virus (family Virgaviridae, genus Tobamovirus) in Nicotiana benthamiana (Rahoutei et al., 2000), and potato virus A and potato virus Y (family Potyviridae, genus Potyvirus) in N. tabacum.

In a review by Alazem and Lin (2017), it was noted that ABA content commonly increases in virus-infected plants as a defense response, and increase in ABA in leaves substantially limit g_s. Virus-induced increases in ABA content have been reported for cucumber mosaic virus in N. benthamiana, bamboo mosaic virus in Arabidopsis thaliana, bamboo mosaic virus in N. benthamiana, and tobacco mosaic virus in N. tabacum (Fraser and Whenham, 1989; Alazem et al., 2014). Therefore, it is possible that the decline in stomatal conductance that we observed in the symptomatic plants might be due to virus-induced increases in ABA synthesis. Additionally, reductions in g_s are a well-known response to multiple wilt-inducing stresses (biotic or abiotic), and stomatal closure due to decreases in leaf turgor limits access to CO₂ by rubisco (Kirkpatrick et al., 1995; Sampol et al., 2003; Farooq et al., 2009; Chastain et al., 2014; Snider et al., 2014; Parkash and Singh, 2020a; Parkash et al., 2021). Furthermore, if g_s reaches sufficiently low levels, even processes that do not necessarily require CO₂ as a substrate (e.g., the thylakoid reactions) will become limited (Medrano et al., 2002). Similarly, in our study, A_n and ETR showed a strong hyperbolic response to g_s (Figure 4). However, when the relative sensitivity of each process to declines in g_s is determined, clear differences in stress sensitivity for carbon assimilation and electron transport are observed. For example, the g_s value required to decrease A_n 50% from the maximum observed value was 0.371molm⁻² s⁻¹ while a 50% decline in ETR from the maximum observed value was observed at a g_s value of 0.022molm⁻² s⁻¹. This indicates substantially greater stress sensitivity for carbon assimilation than electron transport. Under other stresses, where electron transport outpaces carbon assimilation (such as drought), oxidative stress is an inevitable consequence (Lawlor, 2002). Furthermore, anthocyanin production under stress is thought to either attenuate excess solar radiation or to serve a reactive oxygen scavenging role (Close and Beadle, 2003). Thus, the possibility that leaf reddening (Figure 1) in the initial stages of CLRDD is a response to oxidative stress should be evaluated in the future. It is well documented that some plant viruses affect the chloroplasts while infesting the host plant (Bhattacharyya and Chakraborty, 2018), altering the expression of chloroplast- and photosynthesis-related genes (CPRGs), and generating disease symptoms in foliage (Bhattacharyya et al., 2015). For example, tobacco mosaic virus causes a reduction in expression of tobacco chloroplast proteins, reducing photosynthetic efficiency (Lehto et al., 2003). Chloroplasts are also a major source for generating reactive oxygen species (ROS; Ambastha et al., 2015), and ROS play a key role in inducing cell death and subsequently causing leaf senescence under various biotic and abiotic stresses (Van Breusegem and Dat, 2006). Overall, this suggests that CLRDV might have interacted with the chloroplast, thereby contributing to lower photosynthesis rate in diseased plants. Moreover, interaction of the virus with the chloroplast might have increased the production of ROS, which subsequently might have caused the cell death and ultimately leaf senescence in diseased plants.

Our data showed that leaf temperature increased as CLRDD symptoms progressed to more advanced stages (Figures 5–7), where leaf temperature of diseased plants was often higher than air temperature. In addition, plants with more advanced stages of CLRDD symptoms had lower g_s, and declines in g_s limit water loss through the stomatal aperture via transpiration. Transpiration is the dominant mechanism by which plants dissipate excess energy and keep foliage cool (Fitter and Hay, 2012). Therefore, declines in g_s are commonly associated with higher leaf temperature (Chastain et al., 2014; Parkash and Singh, 2020b; Virk et al., 2020a,b; Parkash et al., 2021), as is the case for plants at later stages of CLRDD. Interestingly, while diseased plants have a higher leaf temperature at the hottest part of the day, they also lacked the ability to cool for the majority of the day, even when air temperatures were cooler and solar radiation was lower (e.g., during morning and late afternoon hours). For example, S₂ and S₃ plants had higher leaf temperatures than S₁ or S₀ plants for all sample times after 0900h. Furthermore, at some of the sample times, leaf temperature for S₂ and S₃ plants was at or above air temperature (Figure 7). This is particularly concerning for cotton because the optimal canopy temperature range for metabolic activity is 28±3°C (Burke and Wanjura, 2010). Without the ability to maintain a limited level of homeothermy through transpiration (Mahan and Upchurch, 1988), the effects of plant disease on metabolism are likely only magnified by concurrent exposure to heat stress.

We noted that symptoms of CLRDD described herein (leaf reddening, drooping, increased leaf temperature, and wilting) are also symptoms commonly indicative of bronze wilt (Bell et al., 2002). However, symptoms of bronze wilt often appear during fruit development and no pathogen has been identified as the causal agent; therefore, it was believed to be a physiological disorder associated with specific cotton germplasm tracing to the genetic background of “Tamcot SP-37.” Bronze wilt was particularly prevalent in the 1990s, but the removal of susceptible varieties from the market and avoiding the use of susceptible germplasm in breeding programs have largely eliminated the disease in the United States cotton belt. Nonetheless, it is an insidious disease with an occasional susceptible variety making its way to the market, resulting in localized outbreaks. Because of the substantial overlap in symptomology between bronze wilt and CLRDD, future studies should determine if the two diseases share a common cause.

Plants expressing disease symptoms in the early season stayed extremely stunted throughout the remainder of the growing season, which ultimately limited plant productivity. For example, per plant lint and seed-cotton yield was almost completely eliminated in plants expressing all disease stages (S₁–S₃) in the 2020 season. Among the yield components assessed in the current study, the primary driver of yield loss in diseased plants was a reduction in the number of bolls per plant. Specifically, conspicuously-diseased plants only retained an average of 1–2 bolls per plant, whereas S₀ plants set an average of 19 bolls per plant (Table 2). This observation is in agreement with a recent report that severe disease incidence results in reduced boll set and ultimately reduces seed-cotton yield per plant (Avelar et al., 2020). Field observations of diseased plants suggest that pollen development or shedding may have been compromised but this requires further study. In addition to reductions in boll number, the average boll weight and seed set per boll were also significantly reduced, likely contributing to reductions in per plant lint and seed-cotton yield. Cotton yield losses up to 80% (per unit land area) have been reported due to Blue disease caused by the CLRDV biotype prevalent to South America (Silva et al., 2008). In the current study, seed cotton yield per plant decreased by 99%. Therefore, the future impact of the recently detected North American CLRDV genotype on the United States cotton industry will largely depend on the development of resistant varieties, and if susceptible varieties are planted, the levels of disease incidence which can be attenuated by controlling the cotton aphid vector.

In the current experiment, we also hypothesized that asymptomatic cultivars would exhibit more conservative gas exchange responses (lower g_s) than otherwise healthy-appearing plants of the symptomatic cultivar under field conditions. Our results tentatively support this hypothesis. For example, when evaluated under the same mid-day conditions in 2019, plants of the symptomatic cultivar in the S₀ stage had higher stomatal conductance (g_s) and net photosynthesis (A_n) than either asymptomatic cultivar on the initial measurement date. In 2020, the S₀ stage was found to have significantly higher g_s and numerically higher A_n than an asymptomatic cultivar. There are two possible explanations for our observations. First, healthy plants of the symptomatic cultivar possess innately higher gas exchange rates than asymptomatic cultivars, even in the absence of CLRDV. Recent research has indicated that cotton cultivars exhibit genotypic differences in their gas exchange response to vapor pressure deficit (VPD; Shekoofa et al., 2021). For example, VPD will increase as air temperature increases or relative humidity decreases. Some cultivars will close their stomata to limit gas exchange at high VPD to conserve water and delay wilting. Other cultivars maintain higher gas exchange rates even under hot, dry conditions, making them more susceptible to rapid-onset wilting. Since all plants (normal or with conspicuous disease symptoms) tested positive for the presence of the virus, it was not possible to distinguish these two possibilities. Additionally, the S₀ stage of symptomatic cultivar had higher per plant yields than the asymptomatic cultivar. Thus, in the absence of disease symptoms, higher gas exchange rates in the susceptible cultivar may promote higher yields when compared with asymptomatic cultivars that have more conservative gas exchange rates.

The second possible explanation for our observation is that viral infection induces higher gas exchange rates in otherwise healthy plants by increasing g_s, but only in susceptible cultivars. In a study on potato, it was found that a negative control and virus (potato virus Y) infested plants had similar g_s initially but it decreased in virus infested plants over time (Zhou et al., 2004). Though not due to viral infection, in two rice genotypes, initially g_s increased after pathogen infestation (bacterial blight) then decreased with time in pathogen inoculated plants compared to the negative control, whereas a third genotype had lower g_s in pathogen infested plants during all sampling times after pathogen infestation (Kumar et al., 2013). To elucidate interactions between CLRDV and cultivar for physiological responses to infection will require controlled field experiments in the future that incorporate negative and positive control plants of symptomatic and asymptomatic cultivars.

The objectives of the current study were to determine gas exchange, chlorophyll fluorescence, and leaf temperature responses for a symptomatic cotton cultivar at multiple stages of CLRDD progression and for otherwise healthy-appearing plants of symptomatic and asymptomatic cultivars. The results of our study suggest that symptomatic cultivars exhibiting CLRDD had significant declines in g_s, net photosynthetic rate, and photochemical efficiency of the thylakoid reactions. Photosynthetic electron transport was less sensitive to declines in g_s than was carbon assimilation, and future research should evaluate the possibility that an imbalance between the thylakoid reactions and carbon assimilation leads to oxidative stress in diseased tissues. Another consequence of lower g_s associated with CLRDD was a reduction in the ability to cool through transpiration. This was evidenced by higher leaf temperatures and positive leaf-air temperature differentials for much of the day. Ultimately, the negative impacts of CLRDD on plant physiological processes resulted in near-complete loss of per plant yield in diseased plants. Disease-free plants of the symptomatic cultivar exhibited higher gas exchange rates than the asymptomatic cultivar and had greater per plant productivity. The possibility that conservative gas exchange responses in asymptomatic cultivars promote tolerance to CLRDV should be evaluated further in controlled field experiments. Finally, the relationship between bronze wilt and CLRDD should be investigated.

The original contributions presented in the study are included in the article/supplementary material, and further inquiries can be directed to the corresponding authors.

JS, PC, DS, and SB contributed to the conception and design of the study. JS, PR, DW, SK, and NS were responsible for project execution. VP analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.

This research was funded in part by Cotton Incorporated (grant number 19-114) and by Georgia Cotton Commission.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.