Passion fruit (Passiflora spp.) is a highly important crop with increasing yield levels in recent years. However, its expansion in Brazil faces challenges, including diseases affecting the crop. The main disease is passion fruit woodiness, caused by cowpea aphid-borne mosaic virus (CABMV), which leads to leaf and fruit symptoms, compromising both quality and production. Viral infections directly affect plant physiology and often disrupt gas exchange by impacting leaf function. In this study, our aim was to analyze gas exchange in view of a scale of foliar symptoms caused by CABMV and to estimate genetic parameters to aid in the selection of genotypes with superior physiological performance. Gas exchange measurements were performed on passion fruit leaves from full-sib families showing varying symptoms of infection, as well as on leaves from resistant (P. setacea) and susceptible (P. edulis) individuals. Disease severity was assessed on a rating scale associated with the SPAD index. The estimation of genetic parameters facilitated the determination of variances, heritability and accuracy for each physiological variable examined. Our results confirmed that the presence of CABMV in leaves reduced photosynthetic rates and stomatal conductance, especially in the face of moderate and severe symptoms. Furthermore, genotypic variation played a more significant role in photosynthetic rate, intercellular CO2 concentration and transpiration rate compared to phenotypic variation. Heritability values ranged from moderate to high, and accuracy values were high, validating the reliability of the analyses. In conclusion, the study demonstrates, for the first time, the negative impact of CABMV infection on gas exchange in passion fruit leaves. In addition, it highlights the potential use of physiological variables, such as photosynthesis, in the selection of individuals with superior physiological performance in passion fruit breeding programs.
Research Article
Viral infection by CABMV-cowpea aphid-borne mosaic virus- reduces gas exchange in passion fruit leaves
https://doi.org/10.21203/rs.3.rs-4707997/v1
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Passiflora
photosynthesis
gas exchange
Passion fruit (Passiflora spp.) is a significant crop within the Passifloraceae family, with Brazil alone hosting around 166 species, of which more than 150 are native (Araya et al. 2017; Bernacci et al. 2020). The genus Passiflora encompasses diverse species that are used in breeding programs aimed at enhancing yield, fruit quality, and disease resistance, in addition to reducing production costs. Among these species, Passiflora edulis Sims has gained prominence in the last 30 years due to its economic importance and extensive research (Bernacci et al. 2008).
Brazil stands as a major producer and consumer of passion fruit, with a production of 690,364 thousand tons, harvested area of 46,530 ha, and an average yield of 14,867 kg/ha (IBGE, 2021), suggesting further potential for growth (Carvalho et al. 2021). However, diseases pose a threat to the expansion of passion fruit growing, fruit quality, and crop yield. Since the 1980s, the passion fruit crop has been plagued by diseases and pests that can affect all plant parts. Without adequate control strategies, these biotic factors lead to irreversible losses (Oliveira & Frizzas 2014). Among the diseases, passion fruit woodiness caused by cowpea aphid-borne mosaic virus (CABMV) is a major phytosanitary concern and has been extensively studied in recent years, as it reduces the productive period of the crop and diminishes the commercial value of the fruits (Cerqueira-Silva et al. 2015).
Passiflora edulis is highly susceptible to CABMV (Sacoman et al. 2018), while the related species P. setacea exhibits resistance to the virus (Junqueira et al. 2005; Santos et al. 2015a). A characteristic symptom of the disease is a significant reduction in leaf area, leading to leaf deformations, blisters, wrinkling, and impaired plant growth (Cerqueira-Silva et al. 2014). The virus can also cause thickening of the fruit pericarp, reducing the size and quantity of pulp, which negatively impacts the quality and production of passion fruit (Resende, 2006; Cerqueira-Silva et al. 2014; Sacoman et al. 2018). As the productivity of passion fruit is closely tied to photosynthetic activity, the detrimental effects of CABMV on leaf function are evident, significantly reducing orchard longevity and rendering passion fruit cultivation unsustainable across all production regions (Gonçalves et al. 2021; Santos-Jiménez et al. 2022).
To advance knowledge on the yield of passion fruit, the passion fruit breeding program at UENF (Universidade Estadual do Norte Fluminense) has conducted numerous studies on the effects of biotic factors. These studies have evaluated passion fruit populations derived from interspecific crosses between P. edulis 'UENF Rio Dourado' and P. setacea, as well as backcross generations (Santos et al. 2015b; Santos et al. 2019b; Preisigke et al. 2020a and b; Mendes et al. 2022). The program has also conducted advanced studies on resistance inheritance (Freitas et al. 2015; Santos et al. 2019a). However, research specifically targeting the identification of genotypes tolerant to the virus and associated with photosynthetic capacity has not been conducted. Thus, this study focused on a passion fruit population from the UENF breeding program.
It is worth noting that the presence of the virus in plant tissues directly affects leaf metabolism, particularly gas exchange. For instance, viral infections in papaya leaves have been shown to reduce photosynthetic carbon assimilation, growth, yield, and fruit quality (Singh, 2010). In grapevine leaves (Vitis vinifera L. cv. Malvasia), infection by different viruses can reduce photosynthetic capacity by up to 50% (Sampol et al. 2003), whereas other viruses associated with Vitis leaves decrease not only CO2 assimilation, but also stomatal conductance and transpiration (Endeshaw et al. 2014).
In this context, the general objective of this study was to investigate gas exchange in passion fruit leaves infected by CABMV in different degrees of symptoms. The specific objective was to estimate genetic parameters associated with gas exchange to enable the selection of genotypes with superior physiological performance in the face of viral infection. The hypothesis of this study was directed at confirming whether reductions in foliar gas exchange were related to the severity of symptoms by CABMV infection in passion fruit trees.
Experimental conduct and plant material
The experiment was conducted at the Antonio Sarlo Technical School of Agriculture in Campos dos Goytacazes, a municipality in the northern region of Rio de Janeiro, Brazil (21º74' S, 41º33' W, 11.2 m altitude), within the timeframe of December 2020 to December 2021. Data were collected on three occasions within this period (December 2020, April 2021, and December 2021). The climate in the region is classified as Aw type according to the Köppen-Geiger climate classification, which corresponds to a humid tropical climate with rainy summers, dry winters, and temperatures above 18 ºC in the coldest month (Paes et al. 2012). The average annual temperature in Campos dos Goytacazes is approximately 24 ºC, and the average annual precipitation is 1023 mm (Mendonça et al. 2007).
The plant material used in the experiment was obtained from 90 full-sib families of Passiflora spp. that were part of the UENF intrapopulation selection program aimed at resistance to CABMV. However, for this study, 12 passion fruit plants were selected and marked based on three scale of symptoms (F: few; M: moderate; and S: severe), totaling 36 plants in each experimental design block. In addition to the families, cultivar UENF Rio Dourado (P. edulis) (Viana et al. 2017) was used as control 1 (susceptible), with five plants, and P. setacea was used as control 2 (resistant), with three plants. The experimental design employed was a randomized block design with three replications.
The degree of disease severity was determined using a symptom rating scale with modifications suggested by Oliveira et al. (2013). The scale ranges from 1 to 4, with 1 representing no symptoms; 2, mild mosaic symptoms and no deformations; 3, severe mosaic symptoms and no deformations; and 4, severe mosaic symptoms with leaf deformations. Additionally, the SPAD index, measured using the SPAD-502 chlorophyll meter (Minolta Co. Ltd, Osaka, Japan), was determined for each marked leaf to assess the degrees of infection. For this, 3 leaves per plant were measured, with 10 measurements per leaf, between 8:00 am and 10:00 am, along the leaf blade, avoiding areas with veins, obtaining the mean SPAD index values. Rating scale 1 was not considered in this study due to the absence of symptom-free leaves. Table 1 provides a description of the levels, scale, and index used.
Table 1. Scale of symptoms, rating scale, incidence of CABMV, and SPAD index of the evaluated leaves.
|
Scale of symptoms |
Rating scale |
Disease incidence |
SPAD index |
|
F-Few |
2 |
Mild mosaic symptoms and no deformations |
50.2-54.9 |
|
M-Moderate |
3 |
Moderate mosaic symptoms and no deformations |
39.8-47.5 |
|
S-Severe |
4 |
Severe mosaic symptoms, with leaf blisters and deformations |
23.9-32.4 |
A trellis system with 2.5 m-high posts spaced 4 m apart and wire number 12 at a height of 1.80 m from the ground was used to vertically train the plants in the experimental area. The planting rows were spaced 3.5 m apart. Localized dripping irrigation was employed with a flow rate of four liters per hour, and supplemental irrigation was provided during periods of drought. Liming and fertilizer application followed soil analysis and recommendations by Costa et al. (2008). Cultivation practices were carried out as necessary and in accordance with recommendations for passion fruit crops (Costa et al. 2008).
Climatic data, including relative humidity and maximum, average, and minimum temperatures, were obtained from the National Institute of Meteorology (INMET) database. These data were recorded monthly at the Automatic Station of Campos dos Goytacazes - RJ and evaluated throughout the experimental period (Fig. 1). The maximum, average, and minimum temperatures recorded were 35.6, 25.67, and 20 ºC, respectively, in December 2020; 35.5, 23.64, and 15.9 ºC in April 2021; and 34.9, 24.66, and 17.5 ºC in December 2021. Relative humidity was 77.46, 75.68, and 78.13% in December 2020, April 2021, and December 2021, respectively.
Solar radiation was also recorded during the months of implementing the experiment. The data, initially measured in KJ/m2, were obtained from the INMET automatic station. They were then converted to W/m2 using a conversion factor of 2.02, as described by Mavi and Tupper (2004) and cited by Reis and Ribeiro (2019). Subsequently, the results were further converted to photosynthetically active radiation (PAR) in µmol m–2 s–1, following the formulas 1 and 2.
Monthly measurements of PAR were collected throughout the experiment, specifically during the hours of foliar gas exchange evaluation in the months of December 2020, April 2021, and December 2021. Thus, the average PAR measurement was obtained, which were expressed in relation temperature (Fig. 2a) and relative humidity (Fig. 2b).
Leaf gas exchange evaluations
Leaf gas exchange parameters, i.e. CO2 assimilation rate (A, µmol CO2 m–2 s–1), transpiration rate (E, mmol H2O m–2 s–1), stomatal conductance (gs, mol m–2 s–1), intercellular CO2 concentration (Ci, µmol mol–1), leaf-air vapor pressure deficit (VPDleaf, kPa), and leaf temperature (Tleaf, °C), were evaluated at three time points: December 2020 (383 days after planting - DAP), April 2021 (510 DAP), and December 2021 (745 DAP). The measurements were conducted between 08h00 and 10h00 on completely expanded leaves grown in full sun, positioned on the east face, in the upper middle third of the plant. The LI-6400 infrared gas analyzer (LI-COR, Lincoln, Nebraska, USA) was used for these evaluations. The CO2 concentration inside the analysis chamber was maintained at 400 µmol mol–1, and PPFD at 1500 µmol m–2 s –1 (from a 6400-02B red–blue light source), whereas the temperature and relative humidity inside the chamber were set at 25°C and 60%, respectively.
Data analysis
The data were analyzed using a linear model given by:
in which \(\:{Y}_{ij}\) represents the phenotypic mean of the i-th collection (sampling times) in class (infection severity) j; \(\:\mu\:\:\)is the general constant of the model; \(\:{e}_{i}\:\)is the fixed effect of collection i (i = 1,…,3); \(\:{g}_{j}\) is the random effect of family j (j = 1,…,3) assumed to follow a normal independent distribution with mean \(\:{\mu\:}_{j}\)and variance \(\:{\sigma\:}_{g}^{2}\); and \(\:{\epsilon\:}_{ij}\) is the error term associated with the model, assumed to have a random normal independent distribution with mean 0 and variance \(\:{\sigma\:}_{\mathcal{E}}^{2}\). To assess the homogeneity of the residual variance, Bartlett's test was performed, and the dispersion of the residuals was visually examined in the Cartesian plane. The genetic parameters were estimated based on the mean squares of the effects in the model, including the environmental coefficient of variation (CVe), phenotypic variance (σ2p), genotypic variance (σ2g), environmental variance (σ2e), heritability (h2), intraclass correlation (ICC), genotypic coefficient of variation (CVg), ratio between the genotypic coefficient of variation and the environmental coefficient of variation (CVg/CVe), and accuracy (Ac). Additionally, a Pearson correlation matrix was constructed to assess the relationships between the measured variables and paired data.
For the analysis of the physiological and genetic parameters, the statistical software R (Version 4.1.0) was used. The Origin Pro 2016 software (OriginLab Corporation, Massachusetts, USA) was used to create the graphs and analyze the climatic data and SPAD index.
Determination of the SPAD index
The SPAD index was determined on the three symptom scales (F, M and S) as well as in the susceptible control (P. edulis) and resistant control (P. setacea). The results ranged from 50.2 to 54.9 (F), 39.8 to 47.5 (M), and 23.9 to 32.4 (S). In the control plants, the SPAD indices ranged from 43.8 to 49.4 (P. edulis) and 51.5 to 58.7 (P. setacea).
The evaluated samples differed significantly from each other at the 5% probability level (Fig.3). The mean SPAD indices were 52.93 (F), 43.55 (M), 28.49 (S), 47.04 (P. edulis), and 55.38 (P. setacea) (Fig. 3).
Leaf gas exchange
The mean photosynthetic rates (A)varied between treatments during the three evaluation periods. The lowest A value (4.34 µmol CO2 m–2 s–1) was observed in leaves with a high incidence of the symptoms (S) in December 2021. The highest A values were found in P. setacea, a species resistant to CABMV, with the highest result recorded in December 2020 (19.61 µmol CO2 m–2 s–1) (Fig. 4). In addition, it is possible to observe in Fig.5 how the disease progressed in the plants as a whole and in the leaves over the sampling periods.
The lowest mean stomatal conductance (gs) values were obtained in leaves with moderate (M) and severe (S) incidences of CABMV symptoms, measuring 0.150 and 0.174 mol m–2 s–1, respectively. The highest gs value was observed in December 2021 in P. setacea, reaching 0.562 mol m–2 s–1 (Fig. 4).
Transpiration (E) differed between the three grades of disease symptoms on leaves. The lowest mean was found in P. setacea (1.30 mmol H2O m–2 s–1) in April 2021, whereas the highest E value was detected in P. edulis (3.91 mmol H2O m–2 s–1) in December 2021 (Fig. 4).
Analysis of variance (Table 2) revealed significant effects at the 1% level for the variables A, Ci, and E concerning the three evaluation periods and treatments. For gs, there was only a significant effect observed between treatments. Conversely, leaf-air vapor pressure difference (VPDleaf) and Tleaf showed significant differences between the three evaluation times.
Table 2. Mean squares with degrees of significance of analysis of variance of collection (sampling times) and class (severity of infection) in relation to net CO2 assimilation rate (A, µmol CO2 m–2 s–1), stomatal conductance (gs, mol m–2 s–1), intercellular CO2 concentration (Ci, µmmol m–2 s–1), transpiration (E, mmol H2O m–2 s–1), leaf-air vapor pressure difference (VPDleaf, kPa), and leaf temperature (Tleaf, °C), evaluated in passion fruit.
|
|
DF |
A |
gs |
Ci |
E |
VPDleaf |
Tleaf |
|
Collection |
2 |
677.3*** |
0.01569ns |
63019*** |
16.735*** |
6.429*** |
414.8*** |
|
Class |
4 |
308.1*** |
0.06664* |
10826*** |
3.239* |
0.238ns |
1.9ns |
|
Residual |
238 |
17.0 |
0.02583 |
1983 |
1.305 |
0.130 |
2.1 |
Significant by the F test, with p < .001 '***', p < .01 '**', p < .05 '*', ns: not significant.
The ratios between gas exchanges were analyzed to determine instantaneous water use efficiency (A/E, μmol CO2m–2 s–1/mmol H2O m–2 s–1), intrinsic water use efficiency (A/gs,μmol CO2m–2 s–1/mol m–2 s–1), and carboxylation efficiency (A/Ci,μmol CO2m–2 s–1/µmol mol–1). This analysis revealed significant differences at the 1% probability level between treatment classes for all three physiological ratios and across all three evaluation times (Supplementary Information I).
The mean values of A/E showed significant differences between treatment classes at all three evaluation times. The lowest mean A/E values occurred in P. edulis in December 2020 (4.22 μmol CO2m–2 s–1/mmol H2O m–2 s–1) and December 2021 (1.609 μmol CO2m–2 s–1/mmol H2O m–2 s–1), with a significant difference compared with the other classes. In April 2021, the lowest mean value was observed in S (3.58 μmol CO2m–2 s–1/mmol H2O m–2 s–1). Higher A/E means were found in P. setacea during all three evaluation times (Table 3).
Table 3. Mean values of the ratios between gas exchanges evaluated in passion fruit trees infected by CABMV as a function of treatment classes, in the three evaluation periods. Campos dos Goytacazes - RJ, Brazil. 2021. (F: few; M: moderate; and S: severe), and controls P. edulis (susceptible) and P. setacea (resistant).
|
December (2020) |
|
|||||||
|
Class |
A/E |
A/gs |
A/Ci |
|||||
|
F |
7.48507 |
b |
80.43948 |
a |
0.06502 |
b |
||
|
M |
6.66534 |
c |
66.87855 |
b |
0.05299 |
c |
||
|
S |
6.82748 |
bc |
73.83711 |
ab |
0.04852 |
c |
||
|
P.edulis |
4.22411 |
d |
40.01897 |
c |
0.04914 |
c |
||
|
P.setacea |
17.0106 |
a |
84.3651 |
a |
0.16515 |
a |
||
|
April (2021) |
||||||||
|
Class |
A/E |
A/gs |
A/Ci |
|||||
|
F |
6.62954 |
b |
52.17982 |
b |
0.03492 |
b |
||
|
M |
6.65871 |
b |
55.81971 |
b |
0.03622 |
b |
||
|
S |
3.58927 |
c |
64.96476 |
b |
0.02374 |
c |
||
|
P.edulis |
7.12518 |
b |
51.89003 |
b |
0.04211 |
b |
||
|
P.setacea |
16.68159 |
a |
127.2754 |
a |
0.12221 |
a |
||
|
December (2021) |
||||||||
|
Class |
A/E |
A/gs |
A/Ci |
|||||
|
F |
5.08758 |
ab |
61.71169 |
a |
0.04074 |
b |
||
|
M |
3.75188 |
b |
51.88065 |
ab |
0.02809 |
c |
||
|
S |
3.94839 |
b |
42.95118 |
ab |
0.02675 |
c |
||
|
P.edulis |
1.609 |
c |
25.45181 |
b |
0.04558 |
b |
||
|
P.setacea |
5.74136 |
a |
63.45078 |
a |
0.07868 |
a |
||
* Means followed by the same letter in the column, within the same evaluation month, do not differ from each other, according to the Tukey test, at the 5% probability level.
The lowest mean values of A/gs occurred in P. edulis in December 2020 (40.018 μmol CO2 m–2 s–1/mol m–2 s–1) and December 2021 (25.451 μmol CO2 m–2 s–1/mol m–2 s–1). The highest mean value was found in P. setacea (127.27 μmol CO2 m–2 s–1/mol m–2 s–1) in April 2021. Carboxylation efficiency (A/Ci) had its lowest mean value in S (0.023 μmol CO2 m–2 s–1/µmol mol–1) in April 2021, and the highest mean value was observed in P. setacea (0.16515 μmol CO2 m–2 s–1/µmol mol–1) in December 2020 (Table 3).
Estimation of genetic parameters
The genetic parameters were estimated to assess the genetic structure and environmental influence on the studied population for each of the physiological variables. The results (Table 4) indicate that the environmental coefficient of variation (CVe) was larger than the genetic coefficient of variation (CVg) for gs, E, and VPDleaf. However, for A and Ci, CVg made a greater contribution than CVe.
Table 4. Descriptive statistics and genetic parameters estimated relative to the variables of net CO2 assimilation rate (A, µmol CO2 m–2 s–1), stomatal conductance (gs, mol m–2 s–1), intercellular CO2 concentration (Ci, µmmol m–2 s–1), transpiration (E, mmol H2O m–2 s–1), leaf-air vapor pressure difference (VPDleaf, kPa), and leaf temperature (Tleaf, °C), evaluated in passion fruit.
|
Mean |
CVe |
Min |
Max |
σ2p |
σ2g |
σ2e |
h2 (%) |
ICC |
CVg |
CVg/CVe |
Ac |
||||||||||||
|
A |
9.89 |
41.72 |
0.30 |
23.60 |
102.69 |
97.01 |
5.67 |
94.48 |
85.08 |
99.62 |
2.39 |
0.97 |
|||||||||||
|
gs |
0.22 |
73.96 |
0.01 |
1.15 |
0.02 |
0.01 |
0.01 |
61.24 |
34.50 |
53.68 |
0.73 |
0.78 |
|||||||||||
|
Ci |
273.33 |
16.29 |
71.51 |
379.95 |
3608.56 |
2947.52 |
661.04 |
81.68 |
59.78 |
19.86 |
1.22 |
0.90 |
|||||||||||
|
E |
2.06 |
55.49 |
0.06 |
7.86 |
1.08 |
0.64 |
0.44 |
59.70 |
33.05 |
38.99 |
0.70 |
0.77 |
|||||||||||
|
VPDleaf |
1.08 |
33.51 |
0.48 |
3.05 |
0.08 |
0.04 |
0.04 |
45.42 |
21.72 |
17.65 |
0.53 |
0.67 |
|||||||||||
|
Tleaf |
28.58 |
5.08 |
22.82 |
33.79 |
0.64 |
-0.06 |
0.70 |
0 |
-3.02 |
- |
- |
- |
|||||||||||
CVe: environmental coefficient of variation;Min: minimum value;Max: maximum value; σ2p: phenotypic variance; σ2g: genotypic variance; σ2e: environmental variance; h2: heritability;ICC: intraclass correlation; CVg: genotypic coefficient of variation; CVg/CVe: ratio between the genotypic coefficient of variation and the environmental coefficient of variation; Ac: accuracy.
Genotypic variance (σ2g) had a larger contribution than phenotypic variance (σ2p) for A, Ci, and E (Table 4). In contrast, Tleaf showed a greater contribution from environmental variance (σ2e), which was nearly ten times greater than that of genotypic variance.
Heritability values (h2) ranged from 45.42% (VPDleaf) to 94.48% (A), indicating moderate to high magnitudes. Leaf temperature had a heritability of zero. The intraclass correlation (between the evaluated treatments) showed heritability values ranging from 21.72 (VPDleaf) to 85.08 (A). The ratio between CVg and CVe yielded the highest estimates (2.39, 1.22, and 0.73) for A, Ci, and gs, respectively. Accuracy values ranged from 0.67 (VPDleaf) to 0.97 (A) (Table 4).
Determination of the SPAD index and classification of virus levels
We used the SPAD index to classify the degrees of severity of viral symptoms in each of the leaves used in the experiment. Leaves infected with CABMV exhibited significantly lower SPAD values, while the P. setacea resistant control exhibited higher values. The SPAD index has been widely used as a non-destructive chlorophyll meter (Campbell et al. 1990; Uddling et al. 2007; Ling et al. 2011; Ban et al. 2019). Since chlorophylls are closely associated with the photosynthetic efficiency and growth of plants (Jesus and Marenco, 2008), the reduction in chlorophyll levels due to viral infection by CABMV can be inferred. Moreover, the use of this portable meter serves as a valuable technique for distinguishing healthy leaves from infected ones in research studies.
The S leaves (severe symptoms) displayed lower SPAD values and a more yellowish color, which can be attributed to the presence of the virus and the degradation of chlorophyll, resulting in reduced chlorophyll concentration. The portable chlorophyll meter has also been employed for real-time evaluation of plant nitrogen (N) status, as the intensity of green color is significantly correlated with chlorophyll concentration and leaf N concentration (Gil et al. 2002; Fontes, 2011; Pôrto et al. 2011), given that nitrogen is a crucial component in chlorophyll biosynthesis (Rissler et al. 2002).
Therefore, the measurements conducted using the portable chlorophyll meter allowed for a comparison of symptom development and made it possible to reduce the classification to a subjective assessment, relying on a visual symptom rating scale. Numerous non-invasive technologies and plant detection methods exist which have facilitated the evaluation of pathogen effects on plant physiology (Mahlein et al. 2012).
Gas exchange, climatic factors, and progress of viral infection
The presence of viral infection and the increasing severity of the disease in passion fruit leaves over the evaluation period led to a reduction in leaf gas exchange in the present study. The lowest mean values were observed in leaves with moderate and severe symptoms, indicating that the progression of the virus negatively influenced photosynthetic rate and stomatal conductance. Physiological processes such as photosynthesis and transpiration have long been recognized as sensitive to both biotic and abiotic stresses (Taiz & Zeiger, 2010). Numerous studies have demonstrated that foliar diseases can reduce gas exchange in plants (Chou et al. 2000; Sampol et al. 2003; Pinkard & Mohammed 2006; Berger et al. 2007; Kumudini et al. 2010; Nogueira Júnior et al. 2019; Soni et al. 2022).
Different groups of pathogens, including fungi, bacteria, and viruses, can affect leaf physiology and CO2 fixation rate in infected plants, either reducing, increasing, or not causing any effect (Erickson et al. 2003). Specific to CO2 fixation and metabolic processes, various host-virus interactions have been reported to result in disorders. For example, as the infection of tobacco mosaic virus (TMV) progresses, CO2 assimilation decreases in infected tissues (Takács et al. 2014). Additionally, in spinach, the TMV coat protein, when binding to photosystem 2 (PSII), is considered responsible for the decrease in PSII electron transport (Lehto et al. 2003).
It is noteworthy that in the present study the photosynthetic rate remained high in December 2020, showing no significant differences between the mean values for few (F) and moderate (M) virus symptoms. However, when comparing the same evaluation period at the end of the evaluations in December 2021, with the progression of the virus, a change in the initial observations was observed. There were significant differences between the mean values for moderate (M) and severe (S) virus symptoms compared with the few symptoms (F). The absence of significant differences at the beginning of the experiment is likely attributed to the plants increasing their metabolic processes to provide photoassimilates and energy as a defense against viral infection. Alternatively, it could be a result of pathogen manipulation to exploit plant resources. Similar findings have been reported by Varela et al. (2017) and Alam et al. (2015). According to some reports, viral infection in plants leads to significant reductions in photosynthetic rates, which are correlated with the formation of viral mosaics in leaves (Liu et al. 2014; Shimura et al. 2011).
Most viral infections described in the literature are usually considered capable of inducing a reduction in the photosynthetic rate, which is oftentimes associated with changes in chloroplasts, as in tomato (Montasser & Al-Almy 2015). Turnip yellow mosaic virus (TYMV) causes ultrastructural changes in chloroplasts, interfering biochemically and physiologically with leaf gas exchange (Li et al. 2016). Turnip mosaic virus (TMV) had reduced rates in chlorophyll a, b, and total chlorophyll concentration as the symptoms progressed (Guo et al. 2005), and this reduction in the concentration of these photosynthetic pigments may directly result in a decrease in photosynthetic rates (Sayed, 2003). Maintaining chlorophyll levels is an important factor for chloroplasts to produce energy and reactive oxygen species (ROS), which participate in the counterattack of the plant to virus infection (Liu et al. 2014).
Climatic factors should also be considered as influencers of gas exchange in passion fruit crops. Some authors suggest that temperature and humidity conditions influence gas exchange between plants and the environment (Ferraz et al. 2012; Taiz et al. 2017). The climatic data analyzed in our study indicated that the average temperature and relative humidity did not differ significantly. This means that there were no sudden temperature changes that could have influenced the gas exchange data during the evaluation period. As the passion fruit plant thrives in regions with average monthly temperatures between 21 and 32°C and low relative humidity (Melleti 1996), the data collected and presented in this study support these optimal conditions.
Physiological traits such as transpiration rate and stomatal conductance are directly influenced by environmental factors, particularly temperature and air humidity, which affect the opening and closing mechanism of stomata. Elevated temperatures and low air humidity lead to stomatal closure, resulting in reduced stomatal conductance and transpiration rate. This response serves as a defense mechanism in plants to prevent excessive water loss (Taiz et al. 2017). Stomatal closure also restricts the uptake of CO2 and causes a decrease in intracellular CO2 concentration (Lawlor & Cornic, 2002).
Leaf temperature (Tleaf) is a crucial parameter in gas exchange processes and is closely linked to the water balance of plants. Under thermal stress, plants close their stomata to prevent the loss of water by vapor to the atmosphere, increasing leaf temperature. However, pathogens also influence leaf temperature, and exploring this phenomenon has emerged as a diagnostic strategy for early detection of plant diseases in recent years (Oerke et al. 2006; Barón et al. 2016). Previous studies have reported a decrease in leaf temperature in response to infections by certain pathogens, with values up to 0.8°C lower compared to control samples (Lindenthal et al. 2005; Oerke et al. 2011; Jafari et al. 2016). Consistent with these findings, our study revealed similar results, as plants infected with CABMV and exhibiting advanced disease symptoms exhibited lower Tleaf values compared to the resistant control P. setacea (Table 2).
Furthermore, it has already been shown that the presence of CABMV is one of the factors that affect leaf structure and is associated with functional changes based on anatomical analyzes (Barbosa et al. 2019). These findings revealed that the virus induces changes in epidermal cells, leaf blade thickness, stomatal density per area and parenchyma cells. CABMV is known to be able to induce leaf surface wrinkling (Nayudu 2008) and negatively affect leaf histology, particularly stomatal density, which is positively correlated with gas exchange, as higher density, higher stomatal conductance, thus preventing photosynthesis from being limited in adverse conditions (Justo et al. 2005; Lima et al. 2006).
Water use efficiency (WUE) and instantaneous WUE (iWUE) are indeces that reflects how stomata are regulated during metabolic processes (Manalavan et al. 2009). As in the A/E interaction (WUE), the lowest mean iWUE values were observed in susceptible (P. edulis) and infected (M and S) plants, once again highlighting the influence of viral infection on gas exchange in compromising water use efficiency. It is widely recognized that plants close their stomata to prevent excessive water loss, resulting in reduced gs. Consequently, there is a decrease in E and Ci, which limits the availability of CO2 for photosynthesis (Oliveira et al. 2013). Reductions in conductance mediated by stomatal closure primarily affect transpiration rather than CO2 assimilation due to the resistances involved, with greater resistance for water vapor compared with CO2 (Buckley et al. 2003). It is a parameter of great significance, as it helps identify whether photosynthesis is limited by stomatal constraints or factors associated with stomatal and mesophyll morphology, indicating restricted availability of carbon dioxide for the photosynthetic process, and hampering the biochemistry of photosynthesis (Chaves et al. 2009).
Analysis of carboxylation efficiency (A/Ci, µmol CO2 m–2 s–1/µmol mol–1) revealed significant differences between the treatment groups, with the highest mean values observed in resistant control plants (P. setacea). Once again, the influence of viral infection on all gas exchange ratios, including carboxylation metabolism, was evident, as the lowest A/Ci ratios were detected in infected plants as well as in susceptible control plants (P. edulis).
It is worth noting that the presence of pathogens in leaves can also affect stomatal control. Limitations in stomatal or mesophyll diffusion activities, as well as biochemical and photochemical activities imposed by pathogens, can lead to reductions in photosynthesis (Carretero et al. 2011). Pathogens are capable of inducing alterations in leaf anatomy, as changes in anatomical characteristics and, particularly, stomatal density between plants infected and non-infected by CABMV have been described (Barbosa et al. 2019). Stomatal density is directly associated with gas exchange, with higher density corresponding to greater stomatal conductance and lower photosynthesis limitation (Lima et al. 2006).
Lastly, climatic factors are directly related to the severity of the disease. Temperature, humidity, and precipitation can influence resistance between genotypes (Pinto et al. 2008), as well as virulence and the manifestation of CABMV symptoms. Mendes et al. (2022) demonstrated that lower temperatures and precipitation result in increased virus symptoms from leaves to fruits. Thus, the environment can induce more pronounced symptom development and subsequently reduce gas exchange in plants with a high incidence of CABMV.
Estimation of genetic parameters
The full-sib families of passion fruit studied at the three degrees of severity of viral symptoms exhibited a CVg greater than the CVe for A and Ci, indicating genetic variability in relation to virus symptoms and the physiological variables studied. However, no differentiated variation in response to the three degrees of symptom severity was observed for stomatal conductance (gs), transpiration rate (E), or leaf vapor pressure deficit (VPDleaf), indicating that these variables are more influenced by environmental factors.
The genotypic coefficient of variation is directly related to genetic variance and provides insights into the potential changes that can be achieved through selection in breeding programs. A higher CVg value indicates greater genetic variability (Santos et al. 2016). Additionally, understanding the relationship between CVg and CVe helps identify traits with variation primarily driven by genetic causes rather than environmental factors. A ratio close to 1.0 indicates that genetic causes contribute significantly to the observed variation. Therefore, we can conclude that genetic gains can be achieved in breeding (Hallauer et al. 2010). In this study, high CVg values close to 1.0 were observed for A, gs, Ci, and E, indicating that genetic variability played a dominant role compared with environmental variability.
The observed variability in the results can be attributed to several factors, including the genetic material studied, which encompassed both susceptible (P. edulis) and resistant (P. setacea) plants, as well as families derived from crosses between susceptible and resistant materials. The presence or absence of symptoms of CABMV on the evaluated leaves and the occurrence of heterogeneous leaf coloration also indicated genetic heterogeneity within the genus Passiflora. Carvalho et al. (2021) reported similar variability between resistant and susceptible species and attributed it to the genetic heterogeneity resulting from cross-pollination, which facilitates intense gene flow.
The estimation of variance components revealed that genotypic variance contributed most to the phenotypic variance when analyzing A, Ci, and E. This indicates that the observed variation is primarily due to genetic differences between individuals, with little influence from the environment. These findings also supported the high heritability and accuracy estimates observed. On the other hand, leaf temperature (Tleaf) showed the greatest contribution from environmental variance (σ2e), indicating a stronger influence of environmental factors on this variable.
Variance and heritability are the most relevant genetic parameters in breeding programs (Borém and Miranda 2009). Phenotypic variance is divided into genotypic variance and environmental or residual variance (Templeton, 2011). Understanding phenotypic variability, which results from the interaction between genetic and environmental effects, helps breeders select appropriate selection methods, the locations of test to be conducted on the traits of interest, and replication numbers, as well as to predict genetic gains (Borém and Miranda 2009). However, environmental variations can overshadow genetic variations, making the selection process more complex (Cruz and Carneiro 2006).
Heritability (h2) estimates in this study ranged from medium to high, with the highest value observed for A, whereas Tleaf showed a heritability value of zero. Heritability quantifies the relationship between phenotypic and genotypic values and is an essential parameter for the selection process in breeding for any trait (Cruz and Carneiro 2003). Higher heritability facilitates the identification of individuals suitable as parents for subsequent generations and aids in identifying regions associated with the trait of interest (Viana et al. 2017).
Due to its high heritability, the photosynthetic rate, should be considered as a variable in the selection process for the population of full-sib families of passion fruit under study. However, it is important to note that heritability is not an inherent property of the trait itself but depends on the population and environmental conditions to which it is exposed (Bernardo, 2010).
The accuracy values obtained in this study ranged from moderate to very high, indicating good quality of the experiment according to the classification by Resende and Duarte (2007), which considers accuracy as very high (Acgen ≤ 0.90), high (0.70 ≤ Acgen ≤ 0.90), moderate (0.50 ≤ Acgen ≤ 0.70), or low (Acgen ≤ 0.50). Higher accuracy values indicate greater reliability in individual assessments (Viana & Resende 2014) and enhance the precision of predicting the true genetic variation compared with observed phenotypic variation for each physiological variable. These values confirm that the evaluations and procedures used in this research are highly reliable in predicting genetic values and improving selection accuracy.
In conclusion, this study demonstrates, for the first time, the negative impact of viral infection by CABMV on gas exchange in passion fruit leaves, particularly in individuals with severe mosaic symptoms. Therefore, the physiological parameters can be considered as crucial strategies and evaluation criteria in the passion fruit breeding program.
Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Author Contributions
Conceptualization: Bezerra LBS, Viana AP; Methodology: Viana AP, Campostrini E, Bezerra LBS; Data curation: Bezerra LBS, Bernado WP, Mendes DS; Formal analysis: Silva FA; Original writing and draft: Bezerra LBS. Writing and editing: Campostrini E, Silva FA; Acquisition of financing: Viana AP.
Funding information
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. This study was financed by Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) – Finance Code E-26/010.001275/2015. This study was financed by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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