Elevated Temperature Can Reduce Cucumber Mosaic Virus Transmission in Tobacco Plants by Altering the Insect Vector’s Performance

Article information

Plant Pathol J. 2025;41(4):498-506

Publication date (electronic) : 2025 August 1

doi : https://doi.org/10.5423/PPJ.OA.02.2025.0016

Dineesha N. Balagalla ¹^,^†, Wikum H. Jayasinghe ²^,^†

, Hao Gefei ³, W. M. Wishwajith W. Kandegama ³^,⁴, Jihyun Kim ⁵, Hangil Kim ⁵^,

¹Postgraduate Institute of Agriculture, University of Peradeniya, Peradeniya 20400, Sri Lanka

²Department of Agricultural Biology, Faculty of Agriculture, University of Peradeniya, Peradeniya 20400, Sri Lanka

³National Key Laboratory of Green Pesticide, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Center for Research and Development of Fine Chemicals, Guizhou University, Guiyang 550025, China

⁴Department of Horticulture and Landscape Gardening, Faculty of Agriculture and Plantation Management, Wayamba University of Sri Lanka, Makandura, Gonawila 60170, Sri Lanka

⁵Department of Forest Environment Protection, College of Forest and Environmental Sciences, Kangwon National University, Chuncheon 24341, Korea

^*Corresponding author. Phone) +82-33-250-8364, E-mail) imgksrlf@kangwon.ac.kr

†These authors contributed equally to this work.Handling Editor: Mi-Ri Park

Received 2025 February 1; Revised 2025 May 25; Accepted 2025 June 10.

Abstract

Disease dynamics are significantly influenced by insect vectors through their interactions with viruses and host plants. The objective of this study is to understand how increased temperatures affect virus transmission, providing insights critical for developing climate-resilient pest and disease management strategies. We investigated the effects of temperature on the survival and growth of Myzus persicae (Sulzer) (Hemiptera: Aphididae), a key vector of the cucumber mosaic virus (CMV). Experiments were conducted to assess aphid survival, reproduction, and intrinsic rate of increase on healthy and CMV-infected Nicotiana tabacum plants at 25°C and 30°C. It was observed that higher temperatures did not negatively affect aphid survival. CMV transmission assay was performed by allowing aphids to acquire and inoculate the virus under varied temperature combinations, while the aphid feeding behavior was monitored at different temperatures. The transmission efficiency was markedly reduced at 30°C compared to 25°C, regardless of variations in temperature during virus acquisition and inoculation. Analysis of probing behavior revealed that aphids’ probing behavior differed at 30°C, likely contributing to reduced transmission efficiency at higher temperatures. These findings demonstrate the intricate interplay between temperature, vector behavior, and virus transmission. Together, this study emphasizes the importance of incorporating environmental temperature dynamics into the development of sustainable and climate-resilient strategies for managing vector-borne diseases in agriculture.

Keywords: aphid behavior; climate resilience; cucumber mosaic virus (CMV); temperature effects; virus transmission

Due to global warming, pest and disease pressure can increase including spatial and temporal mismatches between pests and natural enemies, changes in latitudinal and altitudinal distribution, and weakening of crop defence (Moreno-Delafuente et al., 2020). Temperature is a dominant abiotic factor that has direct effects on development, survival, range, and abundance of insects (Bale et al., 2002). As invertebrates are ectothermic and sensitive to temperature changes (Facey et al., 2014), with increasing environmental temperature, insects could uplift development rates, fecundity, and number of generations (Moreno-Delafuente et al., 2020). The temperature should be within the insect thermal range to observe the enhancement of activities and a population boom (Facey et al., 2014). Temperature alters the demographic rates of a few aphid species, and the frequency and amplitude of extreme temperature events were species-specific according to the fitness level (Ma et al., 2015). Fossil evidence of the diversity of leaf damage of the host plant species suggests that insects responded to past climate changes (Bale et al., 2002).

Cucumber mosaic virus (CMV) belongs to the family Bromoviridae, genus Cucumovirus. CMV has wide host range including more than 1,200 species of vegetables, flowers, horticulture crops, and weeds (Scholthof et al., 2011). It is an ubiquitous virus widely distributed in the temperate zone, tropical and subtropical area (Scholthof et al., 2011). Once plants are infected with CMV, it is impossible to cure and ultimately causes quantitative and qualitative yield loss (Karimi et al., 2022).

More than 80 aphid species, belonging to 33 genera, are vectors of the CMV (Scholthof et al., 2011). Myzus persicae (Hemiptera: Aphididae), is a vector of more than 40 plant viruses including CMV (Jayasinghe et al., 2022b). According to the estimations, approximately 50% of insect-borne viruses are transmitted by aphids (Karimi et al., 2022). Aphids can pierce the plant wall, quickly produce a large number of progeny (Karimi et al., 2022) and transmit virus particles in a stylet-borne, nonpersistent manner (Scholthof et al., 2011).

CMV transmission has become a big challenge in agriculture (Karimi et al., 2022), thus understanding the interactions between plant-virus-insect and environmental factors is important to develop sustainable pest management strategies (Jayasinghe et al., 2022b). The expression of virus symptoms of the host plant may differ in increasing temperatures. According to a study based on barley yellow dwarf virus (BYDV) in wheat (Triticum aestivum L.), increasing temperature could trigger earlier and greater expression of virus symptoms (Nancarrow et al., 2014). Other than that, temperature may influence the transmission of insect-vectored disease interactions (Mauck et al., 2014). An increasing rate of aphid activity may result in an epidemic due to prevailing less effective control measures (Scholthof et al., 2011).

Despite the well-established role of aphids in CMV transmission, the influence of temperature on vector behavior and virus spread remains insufficiently understood, especially in the context of climate change. As global temperatures rise, it is critical to determine how these changes impact virus transmission dynamics. Previous studies have shown that elevated temperatures can alter symptom expression and increase disease severity (Nancarrow et al., 2014), but little is known about how temperature affects aphid probing behavior, transmission efficiency, or virus accumulation in the specific CMV–aphid–tobacco pathosystem. Given that virus spread is tightly linked to vector behavior, particularly the frequency and timing of probing events, there is an urgent need to characterise these interactions under varying temperature regimes. This study was therefore conducted to fill this knowledge gap by investigating how temperature influences aphid survival, CMV transmission efficiency, time to first probe, probing frequency, and virus accumulation in both host plants and aphid vectors. Insights from this study will support the development of climate-resilient pest and disease management strategies.

Materials and Methods

Plant, insect stocks, and virus inoculation

M. persicae (Sulzer) (Insecta: Hemiptera: Aphididae) were maintained on Brassica rapa plants as the stock culture. To obtain aphids for different experiments, aphids were transferred and reared on Nicotiana tabacum seedlings grown individually in a 50 mL tube, covered by an air-permeable top. These aphids were raised in growth chambers at 25°C or 30°C. The tobacco seeds were sown in coir dust and at the 3–4 leaf stage, seedlings were transplanted into individual pots. Aphid and plant cultures were grown with 16 h of artificial light, at 24°C. The tobacco plants were inoculated at 4 weeks after planting by rubbing sap of CMV-infected tobacco onto leaves with carborundum. Inoculated plants were grown in growth chambers at 25°C or 30°C. These plants were used as inoculum source plants after 10 days post-inoculation (dpi).

Aphid survival rate

Newly emerged 10 apterous aphids (<1 day old) were introduced individually into a clip cage fixed on a fully expanded 4th or 5th leaf of 8-week-old tobacco plants (healthy, CMV-infected). One clip-cage was set per plant, and the aphids were confined until the end of the survival period. Ten clip-cages (10 replicates) were used. The temperature was kept at 25°C or 30°C in healthy and CMV-infected tobacco plants by maintaining them inside of different growth chambers. Dead adults and new-borne nymphs were removed and counted daily, while the remaining adults were monitored until death. To identify significant differences in aphid survival rates in different treatments, a log-rank test was performed.

Aphid intrinsic rate of increase

Newly emerged apterous aphids (<1 day old) were introduced individually into a clip cage, on a fully expanded 4th or 5th leaf of 8-week-old tobacco plants (healthy, CMV-infected) as one clip cage per plant and confined until the end of the reproduction period. The temperature was maintained at 25°C and 30°C in both healthy and CMV-infected tobacco plants. Emerged nymphs were removed daily and counted until the end of the reproduction period. The pre-reproduction period (d), mean generation time (d/0.738), and intrinsic rate of increase (r_m = 0.738×log₁₀(d / n), as proposed by Wyatt and White, 1977).

CMV transmission bioassay

The effect of temperature change on CMV transmission efficiency was evaluated three times. In all the transmission tests, different steps, including aphid rearing, rearing of the inoculum source plant, rearing of the healthy test plants and virus transmission processes, were conducted at 20°C, 25°C, or 30°C in growth chambers. Aphid mediated virus transmission was conducted as described in Jayasinghe et al. (2021a). In all experiments, aphids reared in 8-week-old healthy tobacco plants, which were grown in growth chambers, were used for the transmission experiment. Adult aphids were starved for 2–3 h and introduced to a fully expanded 4th or 5th leaf of 8-week-old CMV-infected tobacco plant (10 dpi) as an inoculum source. During the transmission step, aphids were allowed to probe and feed for 2 min (acquisition phase) and then transferred onto healthy tobacco seedlings, the test plants, at the two-leaf stage (inoculation phase). The second and the third tests were done following the same conditions except maintaining different combinations of the temperature. For the second test, sets of test plants were maintained at 25°C, and for the third test, both sets of inoculum source plants and the test plants were maintained at 25°C.

Quantitative RT-PCR in plants

To measure CMV levels in CMV-infected tobacco plants a conventional extraction method using TRIzol agent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from the plants grown in 25°C and 30°C temperatures. PrimeScript RT Reagent Kit (Takara, Shiga, Japan) was used for cDNA synthesizing, and PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) was used to perform quantitative reverse transcription polymerase chain reaction (qRT-PCR) in the StepOnePlus Real-Time PCR System (Applied Biosystems). As the reference gene, primer pair 5′-CCACACCTCCCACATTGCTGTCA-3′/5′-CGCATGTCCCTCACAGCAAAA-3′ for amplification of elongation factor 1a gene (EF1a) was used. Primer pair 5′-GTTGACGTCGAGCACCAA CGC-3′/5′-TGGTCTCCTTTTGGAGGCCC-3′ was used for quantification of CMV RNA. For comparison of pathogenesis-related gene 1a (PR1a) expression levels, 5′-GAAGTGGCGATTTCATGACGGCTG-3′/5′-CGAACCGAGTTACGCCAAACCACC-3′ was used. The PCR conditions were as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, 55°C for 15 s, and 72°C for 30 s. By executing qRT-PCR, a data set was obtained followed by data analysis. A two-tailed t-test was performed to compare CMV accumulation/PR1a expression levels in CMV-infected plants grown at 25°C and 30°C temperatures.

Western blot hybridization

The systemic upper leaves of CMV-infected N. tabacum and N. benthamiana plants grown at 25°C and 30°C chambers were collected for western blot analysis at 14 dpi. The total protein was extracted from the infected leaves, and those in the soluble fraction were loaded at 13% polyacrylamide gel via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Coat protein (CP) of CMV was detected by western blot analysis using anti-CP antibodies as described in Kim et al. (2022).

Time for the first probe

The time of the first probing was tested using a visual inspection. It was monitored by observing the activities of the aphid’s antenna and head movements using a Dino-Light Digital microscope (AnMo Electronics Corporation, Taipei, Taiwan). Five-day-old aphids which starved for 2–3 h, were introduced to fully expanded 4th or 5th leaf of 8-week-old CMV-infected tobacco plants. The probing activity of the newly introduced aphids was observed, and the time of the first probe was manually recorded. To determine significant differences in the aphid time of the first probe between 25°C and 30°C temperatures, a two-tailed t-test was performed.

The CMV accumulation in aphids

Apterous aphids (<1 day old) were maintained on healthy tobacco plants. Aphids were starved for 3 h and 6 aphids were introduced into fully expanded 4th or 5th leaf of 8-week-old CMV-infected tobacco plant at 10 dpi, which grown at 25°C and 30°C condition. After 5 min of introduction, the aphids were collected into 2 mL crew cap tubes with two beads (Ø = 4.5 mm) and homogenized with 500 μL TRIzol at 4,000 rpm for 30 s using a Tomy Micro Smash MS-100 (Tomy Seiko, Tokyo, Japan). To extract the total RNA, 100 μL chloroform was added and centrifuged again to get the RNA precipitation. The total RNA was collected by centrifugation and then dissolved in 6 μL of RNase-free water and reverse-transcribed using avian myeloblastosis virus (AMV) reverse transcriptase. The partial ribosomal protein S3 (Rps3) gene from M. persicae was utilized as the reference gene and was amplified using primer pair 5′-CGAGCTGCTCCATCTCGTA-3′/5′-CCCACTTTCCATGATGAATCTCA-3′. Quantitative RT-PCR was performed to detect a difference in CMV accumulation level in aphids between the two samples. Two-tailed t-test was performed for statistical analysis.

Sucrose and glucose levels in phloem sap

At 10 dpi, sucrose and glucose levels were assessed in leaves from four tobacco plants. Samples were analyzed from Healthy-25°C, Healthy-30°C, CMV-infected-25°C, and CMV-infected-30°C. Leaf tissue (0.1–0.2 g) was frozen in liquid nitrogen, ground with a mortar and pestle, and then transferred to 1.5 mL tubes containing 80% ethanol, followed by incubation at 40°C for 18 h. The supernatant obtained after centrifugation at 9,000 ×g for 10 min was placed into clean 1.5 mL tubes, to dryness, and then centrifuged again at 3,000 ×g for 10 min. The resulting supernatant was collected in new 1.5 mL tubes and stored at −20°C until further analysis. To analyze sucrose and glucose levels in the samples, the Boehringer Mannheim Enzymatic BioAnalysis method for sucrose, D-glucose, and D-fructose (R-Biopharm AG, Darmstadt, Germany) was utilized, following the manufacturer’s instructions. To determine if there were differences in sugar levels among the four groups ANOVA was conducted, followed by Tukey’s multiple comparison test.

Results

Probability of aphid survival and development

We first measured the aphid survival on healthy and CMV-infected plants at 25°C and 30°C to investigate the effect of temperature elevation on aphid survival and reproductivity. The log-rank test for the trend showed that there was no significant difference among the aphid survival rates (Fig. 1A), suggesting that the temperature and CMV infection did not seriously affect aphid mortality (log-rank test for trend, P = 0.499).

Fig. 1

Effect of temperature on aphid survival and growth. (A) Probability of survival of aphids in healthy and cucumber mosaic virus (CMV)-infested plants at 25°C and 30°C up to 35 days. Data were mean values (±standard error, SE) (log-rank test for trend, P = 0.499). (B) Each data point represents the Intrinsic rate of increase (r_m) of aphids grown in healthy and CMV-infected tobacco plants grown at 25°C and 30°C. Data were mean values (±SE) (two-way ANOVA with Tukey’s multiple comparison test, n = 10, P < 0.0001; values followed by the same letters were not significantly different at P < 0.05).

Next, intrinsic rate of increase (r_m) was measured to evaluate the effect of temperature on aphid population growth. The average r_m of aphids grown in healthy and CMV-infected tobacco plants at 25°C were 0.01242 and 0.01439, respectively (Fig. 1B). Similarly, the average r_m of aphids grown in healthy and CMV-infected tobacco plants at 30°C were 0.02185 and 0.01358, respectively. Data were analyzed using one-way ANOVA with Tukey’s multiple comparison test showed that the mean r_m was significantly high in aphids grown in healthy tobacco plants at 30°C while other treatments did not show any significant deference. Overall, high temperature increased the aphid reproductivity, but the CMV infection had no significant effect.

Temperature change affects the efficiency of virus transmission by aphids

We then conducted a series of virus transmission bioassays to determine the virus transmission ability of aphids under different conditions. As shown in Table 1, the first experiment, where all the factors were maintained at 25°C, the transmission rate was 73% (Table 1, Exp. 1). When the same experiment was conducted at 30°C, the transmission rate was reduced to 20% (Table 1, Exp. 2). In the experiments 3 and 4, the transmission rates were 66% and 26%, respectively. In the second set of experiments, aphid rearing, test plant rearing and inoculation were done at 25°C while the inoculum plants were maintained at either 25°C or 30°C. As the results, the transmission rates were 73% and 70%, respectively (Table 2, Exp. 1 and 2). In the third set of experiments, we used the inoculum source plant maintained at 25°C only. Experiment 1 gave a transmission rate of 66% (Table 3, Exp. 1), and in experiment 2, where the transmission was conducted at 30°C and all the other factors were maintained at 25°C, the transmission rate was reduced to 20% (Table 3, Exp. 2). Similarly, in the experiment 4 also showed a lower transmission rate of 16% (Table 3, Exp. 4). In the experiment 3 (Table 3, Exp. 3), where inoculation was done at 25°C while all the conditions were same as experiment four, the transmission rate was 60%. Overall, higher temperature conditions (30°C) on the inoculation step significantly reduced the CMV transmission rate.

Table 1

Transmission efficiency of CMV in tobacco plants at 25°C and 30°C

Table 2

Transmission efficiency of CMV in tobacco plants at 25°C

Table 3

Transmission efficiency of CMV in tobacco plants (only the inoculum source plant kept at 25°C were used)

We also checked the virus transmission ability of the aphids at 20°C and 25°C (Supplementary Table 1), to understand how low temperature may affect the transmission. In this experiment, only the temperature during the transmission was changed while keeping temperature at all other steps as 25°C. The results showed reduced transmission efficiency at 20°C compared to that of at 25°C.

Probing behavior of aphids

We then compared the probing behavior of aphids at 25°C and 30°C on tobacco plants (Fig. 2). The mean time for the first probe of aphids at 25°C and 30°C were 58.3 s and 100.5 s, respectively. The mean time for the first probe was significantly different between the two treatments (two-tailed t-test, n = 45, P < 0.0001) (Fig. 2A). We next observed the probing frequency of aphids at two different temperature conditions by measuring total and mean numbers of probing events within the first 5 min of introduction to a tobacco leaf (Fig. 2B). At 25°C, the total number of probing events was 108, with a mean of 1.83 per aphid, whereas at 30°C, the total number of probing events was 66, with a mean of 1.24 per aphid. The Poisson Mean Ratio of probing events per aphid between 25°C and 30°C is approximately 1.64, suggesting that aphids at 25°C have ~1.6 times more probing events than those at 30°C. A statistically significant difference in the number of probing events between the two temperatures were observed (Poisson test, P = 0.014).

Fig. 2

Effect of temperature on probing behavior. (A) The mean (±standard error) time for the first probe was analyzed at 25°C and 30°C (two-tailed t-test, n = 45, ****P < 0.0001). (B) The total and mean number of probing events at 25°C and 30°C temperatures. (C, D) Frequency of probing within first 5 min at 25°C (C) and 30°C (D).

Further, the frequency of probing within the first 5 min revealed distinct two patterns at 25°C (Fig. 2C) and 30°C (Fig. 2D). Of 53 aphids tested at 25°C, there were only three aphids which did not probe while 18 out of 59 aphids did not probe at 30°C (Fig. 2C and D). Majority of aphids probed two times within the first 5 min at 25°C while at 30°C majority of aphids either did not probe or probed only once.

CMV accumulation in plants and acquisition in aphids

We measured the virus accumulation in tobacco plants at 14 dpi. In N. benthamiana, the plants showed comparatively severe symptoms at 25°C than at 30°C (Supplementary Fig. 1A). The symptom development was not visually comparative in tobacco plants at 25°C and 30°C (Supplementary Fig. 1B). CMV accumulation according to western hybridization showed thicker bands in both N. tabacum and N. benthamiana at 25°C compared to 30°C, indicating a significantly higher viral titer in the infected plant grown in 25°C conditions (Supplementary Fig. 2A and B).

Virus accumulation and symptom development were also compared in the infected tobacco plants at 25°C and 30°C at 10 and 24 dpi (Supplementary Figs. 3 and 4). At 10 dpi, mild mosaic symptoms were visible in the systemic upper leaves of the plants grown at 25°C, but the plants at 30°C were asymptomatic (Supplementary Fig. 3A). The CMV RNA levels were nearly seven times higher in tobacco plants maintained at 25°C at 10 dpi (Supplementary Fig. 3B). Similarly, at 24 dpi, the mosaic symptoms were much more severe in the plants grown at 25°C compared to the plants at 30°C, and their CMV levels were nearly 10 times higher at 25°C (Supplementary Fig. 4). However, when we measured the CMV acquisition levels in individual aphids at 25°C and 30°C by qRT-PCR. The mean levels of CMV RNA in aphids did not show a significant difference at 25°C and 30°C (two-tailed t-test, df = 5, P = 0.101) (Fig. 3), thus the viral titer in the inoculum plants did not seriously affect the virus acquisition in aphids.

Fig. 3

The cucumber mosaic virus (CMV) accumulation in aphids in 30°C than in 25°C was not significantly different in 5 min after acquisition feeding (two-tailed t-test, df = 5, P = 0.101). ns, not significant.

Sucrose and glucose levels in phloem sap

The glucose levels did not show any significant difference with the temperature 25°C and 30°C (healthy plants: two-tailed t-test, df = 6, P = 0.666; CMV-infected plants: two-tailed t-test, df = 6, P = 0.750) (Supplementary Fig. 5A). However, the glucose levels were significantly low in CMV-infected plants. The sucrose levels also did not show any significant difference with the temperature 25°C and 30°C and were not affected by CMV infection (two-way ANOVA with Tukey’s multiple comparison test, n = 4, P = 0.3042) (Supplementary Fig. 5B).

Discussion

Insect vectors play a critical role in the transmission and survival of plant viruses, making their interactions with viruses and host plants essential to understanding plant disease dynamics (Jayasinghe et al., 2023). The transmission of plant RNA viruses, such as CMV, depends on interactions between insect vectors and host plants, where the virus often modifies both plant biochemistry and vector behavior to increase its spread (Jayasinghe et al., 2021a). However, elevated temperature can interfere with these virus-vector dynamics through climate-sensitive molecular pathways, impacting the insect vector’s physiology and behavior (Rauf et al., 2024; Roussin-Léveillée et al., 2024). By studying how climate factors, particularly temperature, influence vector-virus-plant interactions, it is possible to establish strategies to develop climate-resilient crops and implements to mitigate virus spread.

In this study, we evaluated how insect vector (aphid) survives and how their population growth was affected by infection of CMV in the host plant at 25°C and 30°C. The survival of aphids at 20 days after introduction was not significant in both CMV-infected and healthy plants at 25°C and 30°C. This showed that the increase of temperature by 5 degrees did not negatively impact the aphid’s survival. Interestingly, the r_m of the aphids grown at 30°C on healthy tobacco plants was significantly higher compared to that of all the other treatments. This indicates that the increase in temperature enhanced the aphid growth, yet the CMV infection did not favour the aphid growth and development at 30°C. At 25°C, in contrast, aphids on both healthy and CMV-infected plants had similar growth and development (Fig. 1).

With this observation, we then tested how different temperatures can affect the onward transmission of the virus through the vector. This was done through a series of virus transmission assays, where we wanted to identify the critical point where temperature affects the virus transmission through aphid vectors. In this experiment, we allowed the aphids to transmit the virus at 25°C and 30°C (acquisition and inoculation steps, respectively) (Table 1). All inoculation experiments were done using 10 dpi inoculum source plants. By the transmission assay conducted at 20°C (Supplementary Table 1), we observed a low level of virus transmission efficiency by the aphid. We also observed very lethargic behavior of the aphid. Since the aphid strain we isolated might have well adopted to survive in tropical regions. Therefore, we mainly focused on 25°C and 30°C for the transmission assay. When the transmission was done at 30°C, the transmission rate was greatly reduced irrespective of the temperatures maintained during the other steps (Table 1, Exp. 2 and 4).

We repeated the same experiment (Table 2) to confirm our observation, where transmission was done at 25°C. We used two inoculum source plants; one maintained at 25°C and the other maintained at 30°C. From CMV quantification analysis, it was evident that the CMV levels in the inoculum source plants maintained at 25°C and 30°C were significantly different at three different time points (Supplementary Figs. 2–4). The decreased CMV levels in 30°C seem to be closely related to two major antiviral responses, salicylic acid (SA)-mediated systemic acquired resistance (SAR) and antiviral RNA silencing. To evaluate the effect of elevated temperature on PR1a expression, the expression levels of PR1a in tobacco grown at 25°C and 30°C. As shown in Supplementary Fig. 6, the elevated temperature significantly enhanced the expression of PR1a, a marker of SAR in tobacco, revealed that the CMV-infected tobacco plants grown on 30°C showed higher level of PR1a expression compared to those on 25°C conditions, suggesting that the enhanced SA-mediated defence responses at elevated temperatures may contribute to the suppression of CMV accumulation. Meanwhile, the increased temperature even affects strength of antiviral RNA silencing by reducing small RNA accumulation (Szittya et al., 2003). Therefore, the temperature-dependent upregulation of PR1a implies that SAR is more effectively activated at 30°C, potentially working in concert with antiviral RNA silencing to limit viral replication and spread in tobacco plants.

However, in both experiments using tobacco inoculum grown at two different temperature showed considerable transmission efficiencies (73% and 70%) (Table 2, Exp. 1 and 2). These results suggest that the viral titer in inoculum plant did not significantly affect CMV acquisition and transmission efficiency of aphid. As explained by Jayasinghe et al. (2021b), this could be due to the saturation of aphid receptor proteins from the CMV particles at a lower concentration. Supporting the hypothesis, the acquisition levels of CMV in aphids were similar and not significantly different between aphids at 25°C and 30°C (Fig. 3). Therefore, we believe that the transmission efficiency in our virus transmission study was mainly determined by the environment temperature in which inoculation was done.

We then repeated the transmission test with inoculum source plants maintained at 25°C only (Table 3) which provided the same inoculum level in the source plant. The results revealed that, despite of the similar viral titer in the source plant when the temperature is five degrees higher (30°C) during the acquisition and inoculation step, the virus transmission efficiency was reduced to 20%, whereas at 25°C, the transmission efficiency was around 70%. Therefore, it was evident that the temperature during the acquisition and inoculation of the virus by the aphid plays a key role in the determination of the transmission efficiency.

To further investigate the possible reasons for the above observation, we experimented with the probing behavior of the aphids at 25°C and 30°C. Probing behavior determines the virus acquisition and transmission efficiency (Fereres and Moreno 2009; Jayasinghe et al., 2022a). The aphids took a significantly longer time to do the first probe when observed at 30°C (Fig. 2A). The number of probing events within first 5 min were also significantly different (Fig. 2B). This indicated that the aphids demonstrated an altered feeding/probing behavior in the tested two different temperatures. We also analyzed the frequency of probing within the first 5 min, which revealed that the probing behavior was different in aphids at 25°C and 30°C (Fig. 2C and D). We believe that this could explain the high level of transmission efficiency resulting at 25°C regardless of CMV titer in the source plant. To justify that the reduced viral titer followed by temperature difference is not critical for virus acquisition of aphids, we measured the virus acquisition levels in aphids. Interestingly, the acquisition of the virus did not show any significant difference (Fig. 3). Therefore, we conclude that the transmission reduction we observed at 30°C was due to the inability to transmit the virus due to modified probing behavior in aphids.

Phloem sap quality is one of the key factors influencing aphid probing behavior, with sugar composition, particularly glucose and sucrose levels, playing a crucial role (Jayasinghe et al., 2023). To assess whether temperature-induced changes in phloem sap quality could explain differences in probing behavior, we measured glucose and sucrose concentrations in both healthy and CMV-infected plants maintained at 25°C and 30°C. The results showed no significant differences in sugar levels between the two temperature conditions (Supplementary Fig. 5). These findings suggest that, in this experiment, the observed variations in aphid probing behavior were not attributed to changes in phloem sap quality.

Although our study did not directly investigate the molecular responses of aphids to elevated temperatures, previous transcriptomic studies in related aphid species such as Aphis gossypii have shown that thermotolerance is regulated by complex gene networks. For example, a study by Liu et al. (2023) revealed that genes such as Hsp70 and CathB play essential roles in thermotolerance. These molecular adaptations may help aphids maintain homeostasis or protect critical physiological functions under thermal stress, directly/indirectly regulating their probing behavior.

Therefore, it is plausible that the behavior change reflects a direct physiological or neurological response of M. persicae to elevated temperatures. Additionally, elevated temperatures may influence aphid gut physiology or salivary gland function, potentially altering virus-vector interactions. While these were not directly measured in this study, they warrant further investigation to fully understand the temperature-dependent dynamics of CMV transmission.

Our findings suggest that increasing temperature from 25°C to 30°C can reduce CMV transmission in tobacco plants, likely due to altered feeding behavior and performance of the aphid vector at elevated temperatures. Understanding how plant-aphid interactions are modified under rising temperatures offers valuable insights into climate-smart pest management strategies. With the impacts of climate change intensifying, incorporating adaptive approaches to manage insect-borne viruses is essential. This knowledge could contribute to resilient agricultural practices, helping to mitigate virus transmission risks in a warm climate and supporting sustainable crop protection as part of climate-resilient adaptation strategies.

Notes

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Acknowledgments

This study was supported by the ‘2024 Research Grant’ from Kangwon National University (Grant No. 202404170001) to Hangil Kim.

Electronic Supplementary Material

Supplementary materials are available at The Plant Pathology Journal website (http://www.ppjonline.org/).

PPJ-OA-02-2025-0016-Supplementary-Fig-1,2.pdf

PPJ-OA-02-2025-0016-Supplementary-Fig-3,4.pdf

PPJ-OA-02-2025-0016-Supplementary-Fig-5,6.pdf

PPJ-OA-02-2025-0016-Supplementary-Table-1.pdf

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Experiment	Conditions^a				Transmission efficiency (infected plants out of 10)			Transmission rate (%)

	A	I^b	T	E^c	Test 1	Test 2	Test 3
Exp. 1	25°C	25°C	25°C	25°C	7	8	7	73
Exp. 2	30°C	30°C	30°C	30°C	2	1	3	20
Exp. 3	25°C	30°C	30°C	25°C	7	6	7	66
Exp. 4	30°C	25°C	25°C	30°C	3	2	3	26