Introduction

Gossypium hirsutum L., the world's most important textile plant, contributes to > 90% of the world’s production of fiber (Cai et al., 2014). In addition, the seeds can be used to extract oil and manufacture bran with high protein content (40–45%) (Alves et al., 2020; De Araújo et al., 2013), which are ingredients in the production of cosmetics, soap, margarine, and animal feed, as well as in the synthesis of plastics and rubber (De Araújo et al., 2013). Despite its importance, cotton cultivation faces numerous challenges, particularly fungal diseases that affect the plant at different development stages (Belot, 2020).

Vascular phytopathogenic fungi can cause wilting or necrosis of plant parts and decrease nutrient uptake due to the obstruction of the conducting vessels (Carmona et al., 2021; Belot, 2020). The most common vascular disease in cotton is Fusarium wilt (Chitarra, 2014; Zambiasi et al., 2017) which is widespread in the agricultural areas of tropical regions. Fusarium wilt is usually associated with infections caused by Fusarium oxysporum (Zhu et al., 2020, 2021) which attacks the roots and causes damping-off (Zaki et al., 2021). Indirect symptoms can be observed on the stems and leaves, which turn yellow between the veins and fall progressively (e.g., Srinivas et al., 2019). Since its first report in 1892, Fusarium wilt has been one of the most destructive diseases in the Cotton Belt in the U.S. (Sanogo & Zhang, 2016), representing the most serious problem faced by cotton crops (Zhang et al., 2021). Similar to F. oxysporum, other species of this genus, such as Fusarium solani, are also known to cause symptoms in cotton plants which act as true hosts (Zhu et al., 2019).

These opportunistic fungal species may trigger direct or indirect symptoms in different plant tissues which may hamper the identification of diseases. Opportunistic fungi take advantage of the nutritional and metabolic imbalances that occur at the different stages of plant development to establish themselves in tissues and interfere with development and productivity (Davis et al., 2006; Kirkpatrick & Rockroth, 2001). In general, opportunistic fungi are ubiquitous species that may be naturally present in the host, that cause symptoms upon infection, and are associated with changes in the symbiont community and dysbiosis (Bass et al., 2019).

Evidence for opportunistic infections caused by Fusarium equiseti in Triticum aestivum L. and Aptenia cordifolia plants is available, with progressive necrotic lesions developing from the roots, wilting, and anatomical damage, thus, causing the death of younger individuals (Lahuf et al., 2018). There have also been reports of damage triggered by F. equiseti on Zea mays L., causing stem rot, indirect damage to reproductive parts, toppling, and premature collapse of the contaminated plants (Swamy et al., 2020). However, in Brassica oleracea var. botrytis L., F. equiseti generates symptoms of leaf wilt and initiates stem rot and root rot, after which the infected plants die within 60 days (Li et al., 2017).

There are few reports on the damage caused by F. equiseti in cotton plants; however, previous studies have shown that Fusarium wilt affects G. hirsutum mainly during the seedling establishment and vegetative stages (Chimbekujwo, 2000). Thus, there is a need to understand the relationship between F. equiseti and G. hirsutum as a function of plant phenology because important metabolic differences occur in cotton plants when they grow from the vegetative to reproductive and finally to the maturation phase (Rehman & Farooq, 2019). Accordingly, in the present study, the hypothesis that cotton plants are differentially physiologically affected depending on the phenological stage and the site of infection (i.e., roots or leaves), when infected by the opportunistic pathogen F. equiseti.

Phytopathogenic fungi are disadvantageous to the host and induce modifications in highly responsive physiological parameters, such as a decline in photosynthetic pigment synthesis, changes in chlorophyll fluorescence, and anatomical damage that is difficult to reverse (see Macioszek et al., 2019; Świderska-Burek et al., 2020; Chhabra et al., 2019). These effects are also known to be triggered by abiotic stress (e.g., Rehman and Azhar, 2021; Van der Westhuizen et al., 2020). Therefore, to test the hypothesis, a greenhouse experiment was developed to control the abiotic factors, promote artificial infection, and ensure that the observed physiological and histopathological responses were solely due to the biotic stress caused by the attack of the phytopathogenic fungus.

In situations of biotic stress, even before the appearance of symptoms, alterations in the photochemical parameters and gas exchange serve as evidence of damage. In most cases, pathogens can affect photosynthesis from lesions caused in the chloroplasts. Moreover, during infection, fungal effectors are specifically targeted toward the chloroplasts (Kretschmer et al., 2019) given the central role of this organelle in plant immunity through the synthesis of secondary metabolites, defense compounds, and phytohormones, such as jasmonic acid and salicylic acid (De Torres Zabala et al., 2015; Lu & Yao, 2018; Sowden et al., 2018). These attacks on chloroplasts can affect light energy utilization and incur productivity losses (Lu & Yao, 2018).

This work also sought to unravel the mechanisms by which F. equiseti interacts with the epidermal structure of G. hirsutum during the infectious process. Some phytopathogenic fungi develop typical structures of hyphal penetration, such as appressoria and transpressoria, in the region of contact with the host cells (Ryder et al., 2022; Cruz-Meireles et al., 2021) or use classical entry gates, such as stomata or lenticels, keeping them open through the production of phytotoxins and/or effectors (Navarro et al., 2022; Nemesio-Gorriz et al., 2019; Wu & Liu, 2022). Accordingly, a description of the histopathological mechanisms involving plant-phytopathogen association can assist in the development of management and biotechnological techniques applied to the prevention and/or control of diseases. Thus, the objective of this study was to evaluate the physiological responses and histopathological mechanisms involved in the interaction of cotton plants with the opportunistic phytopathogen F. equiseti. The focus was the most aggressive route of infection (roots or leaves) and the most susceptible developmental stage of the plant (vegetative, reproductive, or maturation).

Materials and methods

Isolation and identification of the phytopathogenic strain

Symptomatic leaves of G. hirsutum presenting necrotic damage and wilt were randomly sampled in a cotton crop grown in the rural area of Primavera do Leste municipality, MT, Brazil (15°23′12 "S and 45°23′12W; 650 m altitude). Four samples were collected (four infected plants). The leaves were placed in aseptic plastic bags and transported to the Agricultural Microbiology Laboratory of the IFGoiano campus Rio Verde, where they were subjected to superficial asepsis for the removal of epiphytes. To achieve this, the leaves were immersed in a 2% Tween 80 solution and agitated at 150 rpm for 5 min. Then, the leaves were washed with water and disinfected using 70% alcohol for 1 min, sodium hypochlorite for 3 min, and 70% alcohol for 30 s, and finally rinsed again three times using sterilized distilled water.

To obtain the phytopathogen, 1 × 1 cm fragments were taken directly from the necrotic or wilted region of the leaf and deposited in solid potato dextrose agar (PDA) medium (an infusion of 200 g of potato, 20 g of dextrose, and 15 g of agar) in Petri dishes. The dishes were incubated at 35 °C for 3 days, when vegetative mycelium was observed to emerge through some of the fragments. Fragments of these mycelia were isolated, purified in PDA, and subsequently identified.

For identification, the genomic DNA of a specimen was extracted in triplicate based on the procedure of Cheng and Jiang (2006) using a Miniprep extraction kit (Axygen Biosciences, USA), according to the manufacturer’s instructions. Identification was performed via partial sequencing of the internal transcribed spacer, and calmodulin and β-tubulin genes after amplification and purification, thus, generating sequences with an average length of 520, 455, and 350 base pairs, respectively. Sequencing was performed using the Sanger method. For phylogenetic inference, the sequences were paired via their similarity with the sequences from GenBank via BLASTn (Altschul et al., 1990), using a homology of > 97%. The sequences were then concatenated and aligned with the homologous sequences extracted from GenBank for seven Fusarium species (F. equiseti – 597 and 605 bp, F. camptoceras – 576 bp, F. clavum – 698 bp, F. cf. incarnatum – 1,228 bp, F. ipomoeae – 689 bp, F. lacertarum – 580 bp, and F. solani – 552 bp), using the Clustal Omega software (Sievers & Higgins, 2014).

Selection of the evolutionary model of the sequences was performed using the Bayesian information criterion implemented in jModelTest 2 (Darriba et al., 2012). The model K80 was selected and the phylogenetic trees were inferred using the methods based on Bayesian inference in MrBayes v.3.2.6. (Ronquist et al., 2012). For each tree, four independent runs were performed by assigning 10 × 106 generations to the chains and sampling the a posteriori probability distribution every 500 generations. Prior to calculating the consensus tree, and to ensure convergence of the chains, the first 2,500 sampled trees were discarded. The phylogenetic tree with the highest Bayesian likelihood was visualized and edited using FigTree v 1.4.4 (Rambaut, 2014). Sequences from the Penicillium chrysogenum species were used as the outgroup.

Plant material and inoculation

Experiments were conducted using cotton seeds of the FMT 910 cultivar not treated with fungicides or insecticides. Seeds were grown in 5-L pots containing 7.5 kg of soil. The soil was previously analyzed (Table S1) according to the methodologies proposed by EMBRAPA (2017). The chemical and nutritional attributes were corrected with macro and micronutrients according to the Goiás Soil Fertility Commission (CFSG, 1988).

Two seedlings were grown per pot. The plants were placed in a greenhouse, and the atmospheric conditions were monitored daily. During the cultivation cycle, the temperature and humidity varied between 28 °C and 36 °C, and 30% and 48%, respectively. The average photosynthetically active radiation (PAR) and CO2 concentration was between 598 and 1,011 μm, and 575 and 662 ppm, respectively. The plants were irrigated daily as required.

The mycelia of F. equiseti were cultivated in Luria Bertani medium (tryptone, 10.0 L–1; yeast extract, 5.0 L–1; sodium chloride, 5.0 L–1; pH 7.0 ± 0.2) for 7 days at 30 °C. Healthy plants were inoculated with 5-mm mycelial fragments by extracting them directly from the dishes and depositing them in contact with the plant tissues. In the foliar inoculation treatment, the mycelial fragments were deposited on the adaxial side of the leaf, and five leaves per plant were inoculated (counting downwards from the apical leaf of each plant). In the root inoculation treatment, the fragments were deposited in 5-cm deep grooves made in the rhizosphere region attached to the main root. Five grooves were made around the root of each plant. These inoculations were programmed to occur in three phenological stages: vegetative, reproductive, and maturation. In the vegetative phase, the plants were inoculated with the phytopathogen in V2 stage (phenological stage that begins when the second leaf reaches 2.5 cm in length and ends when the third leaf reaches 2.5 cm in length). The reproductive phase was considered when all plants presented flowers or developed floral buds, and the maturation phase was considered when all plants presented bolls. The results of the induced exposure to the phytopathogen were compared to those observed in non-inoculated control plants.

The experiment was conducted in randomized blocks using a double factorial scheme (2X3 + 1), i.e., involving two types of infection of the phytopathogen (foliar and root), three phases of cotton development (vegetative, reproductive, and maturation) and a control treatment consisting of non-inoculated plants. The control plants were also evaluated in the three phases of plant development. Each treatment was evaluated in five replicate experiments with two plants per pot in each replicate.

Level of photosynthetic pigments

All physiological analyses were performed by sampling the second expanded leaf, counting downwards from the apical leaf. This leaf was inoculated in the foliar inoculation treatments and not inoculated in the root inoculation treatments. The photosynthetic pigments were evaluated by obtaining three 5-mm diameter leaf discs from all the samples. When the leaf was inoculated, discs were taken from the leaf region farthest possible from the site of infection. The discs were incubated in amber flasks in a solution of dimethyl sulfoxide saturated with CaCO3. The flasks were incubated in a water bath (TECNAL, Banho Dubnoff, TE-053) at 65 °C for 8 h under agitation at 30 rpm for pigment extraction. The absorbance of the extract was read using a spectrophotometer (SHIMADZU, UV-1800 UV–visible spectrophotometer) at 665, 649, and 480 nm corresponding to chlorophyll a, chlorophyll b, and carotenoids, respectively. The concentrations of these pigments were measured according to Wellburn (1997).

Chlorophyll a fluorescence

The OJIP transient fluorescence of chlorophyll a was determined using a FluorPen FP 100 portable fluorometer (Photon Systems Instruments; Drasov, Czech Republic). The inoculated leaves of the sample units were previously exposed to the dark for 30 min to complete the oxidation of the photosynthetic electron transport system. Subsequently, the samples were subjected to a pulse of 3,000 µmol m–2 s–1 blue light. The minimum fluorescence (F0) was measured at 50 μs when all the PSII reaction centers were open; this was defined as step O which was followed by step J (at 2 ms), step I (at 30 ms), and step P (when maximum fluorescence (FM) occurred when all PSII reaction centers were closed). These values were used to estimate various bioenergetic indices of PSII according to Strasser et al. (2000). The values for the specific light absorption flux per reaction center (ABS/RC), captured energy flux per reaction center at t = 0 (TR0/RC), electron transport flux per reaction center (ET0/RC), specific energy dissipation flux at the level of the chlorophylls of the antenna complex (DI0/RC), photosynthetic performance index (PIABS, which incorporates the processes of the energy cascade from the first uptake events to the reduction of PQ), maximum quantum yield of primary photochemistry (PHIPO), probability of an exciton moving an electron through the electron transport chain after the quinone (PHI0), and quantum yield of electron transport (PHIE0) after adaptation of the leaves to the dark (30 min) were estimated.

Gas exchange

Gas exchange was assessed at 08:00 using an IRGA infrared gas analyzer coupled to a fluorometer (model LI-68400XT, LI-COR Inc., Lincoln, USA). Data were obtained for the inoculated leaves using PAR (1,000 μmol photons m–2 s–1), a block temperature of 25 °C, and relative humidity of ~ 50%. The net photosynthesis rate (A; µmol of CO2 m–2 s–1), stomatal conductance (gs; mol of H2O m–2 s–1), transpiration (E; mmol of H2O m–2 s–1), internal carbon concentration (Ci; µmol CO2 mol–1), water use efficiency (WUE; µmol CO2 mmol H2O s–1), and instantaneous carboxylation efficiency (A/Ci) were measured.

Scanning electron microscopy

Leaf histopathological evaluations were performed using images obtained using scanning electron microscopy. Leaf fragments (1 cm2) were prefixed with Karnovsky solution, post-fixed with osmium tetroxide, dehydrated in ethanol, transferred to amyl acetate, and dried to the critical point in a CO2 dryer. The samples were then coated with gold using an ion jet and images were obtained using a scanning electron microscope (Jeol JSM-IT300LV, JEOL USA, Inc., Peabody, MA, USA). These analyses were performed only on plants inoculated through the leaves with samples collected 10 days after inoculation.

Statistical analysis

The data observed for the different physiological parameters were subjected to a normality test and ANOVA. For this, the means observed for each replicate were compared independently within the developmental phases and types of inoculation and in the interaction phases × type of inoculation. When the effect of the exploratory variables (type of infection and developmental phase) was verified, the mean values were compared using the Tukey test at 5% significance. To better understand the relationship between the exploratory variables and physiological variables, these were analyzed together using a correlation matrix and combined in a principal component analysis (PCA). As these variables have different measurement units, correlation PCAs were performed and constructed using data standardized to mean = 0 and standard deviation = 1. The number of components was chosen based on the eigenvalues (> 1.0) and explained variance (> 80%). All analyses were performed using R 4.2.1 software (R Core Team, 2021).

Results

Identification of the phytopathogen

The recovered phylogeny identified the phytopathogenic strain as F. equiseti based on its similarity with the MZ921525.1 strain. The two strains in question constitute a distinct clade, strongly supported and genetically distant from other Fusarium species, such as F. cf. incarnatum, F. camptoceras, and F. solani (Figure S1).

Photosynthetic and photochemical physiological responses in the interaction

The mean chlorophyll a levels in the leaves were affected by the developmental phase of the plants (p = 0.000); hence, the lowest mean values were observed at the maturation phase (Fig. 1a). The interaction developmental phase × type of inoculation (p < 0.001) also had an effect, with the highest mean values in the vegetative phase being observed in the non-inoculated plants and the lowest values being observed in the leaves of inoculated plants. In the reproductive phase, the highest mean values were observed in the non-inoculated and leaf-inoculated plants, and the lowest values were observed in the root-inoculated plants.

Fig. 1
figure 1

Levels of the photosynthetic pigments chlorophyll a (a), chlorophyll b (b), chlorophyll a/b ratio (c), and carotenoids (d) observed in Gossypium hirsutum plants subjected to Fusarium equiseti infection in the leaves or roots in three phenological stages (vegetative, reproductive, or maturation). The letters above the frame indicate the statistically significant differences between phenological stages, and the letters above the bars compare the types of inoculation (leaf or root) and non-inoculation, within the same phase. Comparisons were made using the Tukey test at 5% significance

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For chlorophyll b, the effect during the developmental phase (p = 0.000) was similar to that for chlorophyll a; plants in the maturation stage showed the lowest mean values for chlorophyll b (Fig. 1b). There was a significant interaction of the developmental phase and type of inoculation (p < 0.001). In the vegetative phase, the highest mean values of chlorophyll b were observed in the healthy, non-inoculated plants, and the lowest values were observed in the inoculated plants in the leaves. In the reproductive phase, non-inoculated plants showed the highest mean levels of chlorophyll b and no differences between the types of infection.

The different phases of development influenced the chlorophyll a/b ratio (p = 0.000) with the highest mean values in the maturation phase, indicating a significant reduction in chlorophyll b content in relation to chlorophyll a production (Fig. 1c). In the vegetative phase, the highest values for this relationship were observed in the non-inoculated plants, whereas in the reproductive phase, non-inoculated plants and those inoculated in the leaves presented the highest mean values for the chlorophyll a/b ratio. However, no differences were observed in the ratios in the plants inoculated using different routes in the maturation phase.

Regarding the carotenoid levels in the leaves, the mean values were affected by the developmental phase (p = 0.000) similar to that observed for chlorophyll a and b, i.e., the lowest values were obtained in the plants sampled in the maturation phase (Fig. 1d). However, the types of infection also affected the mean values observed (p = 0.021) with the non-inoculated plants exhibiting the highest levels of these pigments (12.25 µg cm–2), followed by those inoculated in the aerial part and root (11.42 and 11.06 µg cm–2, respectively). The interaction developmental phase × type of inoculation (p = 0.000) also had an effect, with the behavior being identical to that verified for the chlorophyll a/b ratio, i.e., the non-inoculated plants presented the highest levels of carotenoids in the vegetative phase. In the reproductive phase, the highest means were observed in non-inoculated plants and those inoculated through the leaves, whereas in the maturation phase, there were no differences observed between the means obtained for the different types of infection.

The photosynthetic rate of cotton plants was affected by the developmental phase (p = 0.000); in the maturation phase there was a significant decrease in the mean values associated with this parameter (Fig. 2a). The interaction developmental phase × type of inoculation affected the photosynthetic rate (p = 0.010). In the vegetative phase, the photosynthetic rate was reduced. In the reproductive phase, there were no differences between the mean values observed for different types of inoculation. However, in the maturation phase, photosynthesis was affected in the plants inoculated through the roots.

Fig. 2
figure 2

Photosynthetic rate (A) (a), internal concentration of CO2 (Ci) (b), and A/Ci ratio (c) observed in Gossypium hirsutum plants subjected to Fusarium equiseti leaf or root infection in three phenological stages (vegetative, reproductive, and maturation). The letters above the frame indicate the statistically significant differences between phenological stages and the letters above the bars compare the types of inoculation (leaf or root) and non-inoculation, within the same phase. Comparisons were made using the Tukey test at 5% significance

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In contrast, the internal concentration of CO2 was not affected by the developmental phase, but by the type of inoculation (p < 0.001) (Fig. 2b). Thus, plants inoculated through the roots and leaves exhibited the highest mean Ci (349.50 and 330.96 µmol CO2 mol–1, respectively). Similarly, the A/Ci ratio was affected by the type of inoculation (p = 0.017) with the non-inoculated plants presenting the highest mean value (0.20) (Fig. 2c), which was clearly impacted by the low Ci and high A values of these plants.

The stomatal conductance was affected by the cotton developmental phases (p = 0.000) with plants in the vegetative phase exhibiting higher conductance than those in the reproductive phase. However, a drastic reduction in gs was identified in plants in the maturation phase (Fig. 3a). The types of inoculation also affected the gs (p = 0.000), thus, plants infected through the leaves and roots showed higher conductance (0.82 and 0.80 mol H2O m–2 s–1, respectively) than plants in the control, non-inoculated treatment group (0.26 mol H2O m–2 s–1). The interaction developmental phase × type of inoculation affected stomatal conductance (p = 0.006); thus, in the vegetative and reproductive phase, the non-inoculated plants exhibited the lowest stomatal conductance values, whereas in the maturation phase, no difference was observed between the means obtained for the different infection routes.

Fig. 3
figure 3

Stomatal conductance (gs) (a), transpiration rate (E) (b), and water use efficiency (WUE) (c) observed in Gossypium hirsutum plants subjected to Fusarium equiseti leaf or root infection in three phenological stages (vegetative, reproductive, and maturation). The letters above the frame indicate the statistically significant differences between phenological stages and the letters above the bars compare the types of inoculation (leaf or root) and non-inoculation within the same phase. Comparisons were made using the Tukey test at 5% significance

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The developmental phases influenced the transpiration rate. Plants in the vegetative stage showed higher transpiration rates than those in the reproductive and maturation stage (p = 0.000) (Fig. 3b). The type of inoculation also affected the parameter E (p < 0.001). Plants inoculated through the roots and leaves showed higher mean E values (13.12 and 10.31 mmol H2O m–2 s–1, respectively) than the non-inoculated plants (7.76 mmol H2O m–2 s–1). When the interaction developmental stage × inoculation type (p = 0.021) was investigated, differences appeared only in the plants sampled at the reproductive stage. At this phase, the non-inoculated plants showed the lowest mean E values.

In contrast, WUE was not affected by the developmental stages (Fig. 3c), but was affected by the type of infection (p = 0.000) and the interaction developmental stage × type of inoculation (p = 0.040). Non-inoculated plants exhibited the highest WUE (2.00 µmol CO2 mmol H2O–1) followed by plants inoculated through the leaves (1.40 µmol CO2 mmol H2O–1) and those inoculated through the root (0.91 µmol CO2 mmol H2O–1). In the vegetative as well as reproductive stages, the highest mean WUE was observed in the non-inoculated plants, whereas in the maturation stage, non-inoculated plants and those infected through the leaves showed the highest mean WUE values.

The developmental stages of cotton influenced the primary photochemistry (p = 0.037), such that plants in the maturation stage exhibited higher mean ABS/RC values and those inoculated through the leaves showed the lowest mean values (Fig. 4a). The inoculation method did not affect primary photochemistry, but when the inoculation types were split within the phases (p = 0.032), the non-inoculated plants in the vegetative stage exhibited slightly lower ABS/RC values than the infected plants. In the maturation stage, the inoculated plants also exhibited lower ABS/RC values with the root-inoculated plants showing the highest mean values. The ET0/RC was also affected by the developmental phase of the plants (p = 0.000). Plants in the maturation stage exhibited higher mean values for this parameter (Fig. 4b). Regarding the types of inoculation in the different phases, differences were observed only in the reproductive phase, with plants inoculated in the root system being more affected by this parameter.

Fig. 4
figure 4

Absorption flux per reaction center (ABS/RC) (a), electron transport per reaction center (ET0 /RC) (b), energy flux per reaction center at t = 0 (TR0 /RC) (c), and dissipated energy flux per reaction center (DI0/RC) (d) observed in Gossypium hirsutum plants subjected to Fusarium equiseti leaf or root infection in three phenological stages (vegetative, reproductive, and maturation). The letters above the frame indicate the statistically significant differences between phenological stages and the letters above the bars compare the types of inoculation (leaf or root) and non-inoculation within the same phase. Comparisons were made using the Tukey test at 5% probability

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The developmental phase affected TR0/RC in a similar way to ET0/RC (p = 0.000). Thus, plants in the maturation phase exhibited the highest mean values for this parameter (Fig. 5c). An effect due to the interaction developmental phase × inoculation type (p = 0.026) was observed, with the plants sampled in the reproductive phase showing higher means for TR0/RC when inoculated in the leaves; the plants in the maturation phase inoculated in the leaves or roots exhibited high TR0/RC values when compared to the non-inoculated plants. The DI0/RC was affected by the developmental phase (p = 0.032) with plants in the maturation phase being more affected (Fig. 4d). Regarding the types of inoculation, the non-inoculated plants exhibited lower energy dissipation values in the vegetative phase, whereas non-inoculated plants and those inoculated through the roots showed the lowest means in the reproductive phase. In contrast, plants inoculated through the roots exhibited higher mean DI0/RC values despite the non-inoculated plants having dissipated less energy in the form of heat.

Fig. 5
figure 5

Photosynthetic performance index (PIABS) (a), maximum quantum yield of primary photochemistry (PHIPO) (b), probability that a trapped exciton moves an electron into the electron transport chain beyond Quinone (Qa) (PHI0) (c), quantum yield of electron transport, (PHIE0) (d) observed in Gossypium hirsutum plants subjected to Fusarium equiseti leaf or root infection in three phenological stages (vegetative, reproductive, and maturation). The letters above the frame indicate the statistically significant differences between phenological stages, and the letters above the bars compare the types of inoculation (leaf or root) and non-inoculation within the same phase. Comparisons were made using the Tukey test at 5% significance

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The photosynthetic yield index (PIABS) was influenced by the developmental stage of cotton (p < 0.001), with plants in the reproductive stage exhibiting the lowest yields (Fig. 5a). Similarly, the type of inoculation also affected the photochemical yield (p = 0.015) and, overall, the non-inoculated plants showed a better performance (0.60) followed by the root- (0.33) and leaf (0.27)-inoculated plants. In the vegetative and maturation stage, the non-inoculated plants showed a better performance, whereas in the reproductive phase, the highest mean values for this parameter were observed in non-inoculated plants and those infected through the roots. PHIPO was affected only by the interaction developmental phase × type of inoculation (p = 0.040). Thus, non-inoculated plants exhibited higher mean PHIPO values in the vegetative and maturation phase. However, no differences were observed between the mean values obtained for the different types of F. equiseti inoculation in the reproductive phase.

The developmental stage affected PHI0 (p = 0.000), with plants presenting the lowest means in the reproductive phase, followed by the vegetative and maturation phase (Fig. 5c). The types of inoculation also affected PHI0 (p = 0.023). Non-inoculated plants exhibited the highest PHI0 values (0.33), followed by the root- (0.25) and leaf (0.23)-inoculated plants. When the interaction developmental phase × type of inoculation was evaluated (p = 0.040), the non-inoculated plants and those inoculated through the leaves performed better in the vegetative phase. Similarly, in the maturation phase, the performance of non-inoculated plants was superior. However, there were no differences observed between the mean values obtained for the different types of infection in the reproductive phase.

The quantum yield of electron transport (PHIE0) was affected by the phase (p = 0.000), with the behavior being similar to that verified for PHI0, i.e., the plants in the reproductive stage exhibited the lowest mean values, followed by plants in the vegetative and maturation stage (Fig. 5d). The types of infection also affected the mean PHIE0 values (p = 0.025) with the non-inoculated plants exhibiting the highest values (0.24), followed by the root- (0.17) and leaf-inoculated plants (0.16). When assessing the types of infection in the developmental phases, the plants inoculated through the roots and leaves were equally affected in both the vegetative and maturation phase, exhibiting lower yields than the non-inoculated plants. There were no differences between the mean values in the reproductive phase.

The PCA of variables, in relation to the developmental stages, showed that the differences between the maturity stage and other stages were defined by the high values recovered by the chlorophyll a/b ratio and high mean values observed for the photochemical stress indicator variables (ABS/RC, ET0/RC, TR0/RC, and DI0/RC) (Fig. 6a). Moreover, the difference observed between the plants in the reproductive stage and those in the other stages was defined by the high WUE. However, the high A, E, gs, and A/Ci values were more related to the plants in the vegetative stage.

Fig. 6
figure 6

Analysis of the main component of photosynthetic pigment levels, gas exchange, and chlorophyll a fluorescence in Gossypium hirsutum plants subjected to Fusarium equiseti infection at three phenological stages (vegetative, reproductive, and maturation) (a) with the infection initiating at the leaves or roots (b). Chla, chlorophyll a; Chlb, chlorophyll b

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Regarding the types of inoculation, the control plants displayed high mean levels of chlorophyll a and b and carotenoids (Fig. 6b). Additionally, the mean values observed for the photochemical yield parameters (PIABS, PHIPO, PHI0, and PHIE0) and WUE were higher in these plants. However, the root-inoculated plants showed high photochemical stress values (ABS/RC and DI0/RC) and high transpiration rates (E).

Foliar histopathology

At all stages of cotton development, histopathological examinations revealed effective hyphal development along the leaf lamina (Fig. 7a) and spores in germination. A frequent association of the hyphae with the contact region of the dorsal wall of the stomata and adjacent epidermal cells was also observed (Fig. 7b, d, and e), and was not verified as direct penetration via the stomatal pore, even in open or partially open stomata. Thicker hyphae showed regions of segmentation into thinner and more numerous hyphae (Fig. 7c), but no appressoria formation was observed in this region of the epidermal surface.

Fig. 7
figure 7

Microscopic aspects of Fusarium equiseti leaf colonization on the adaxial surface of Gossypium hirsutum leaves observed in three phenological stages (vegetative, reproductive, and maturation). The development of hyphae (a and c) and interaction of hyphae with cells adjacent to stomata (b, d, and e). In d and e, the arrows indicate the region of penetration of the hyphae into the tissue

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As cotton leaves present numerous glandular trichomes in their epidermis (Fig. 8a), surprisingly, the hyphae of F. equiseti were constantly interacting with the cells that constitute these trichomes, especially in the basal region (Fig. 8b and c); where not only hyphae but also asexual reproduction structures, such as phialides and microconidia, were observed (Fig. 8b). Ornate surface leaf trichomes were also associated with colonizing hyphae (Fig. 8d).

Fig. 8
figure 8

Microscopic aspects of Fusarium equiseti leaf colonization on the adaxial surface of Gossypium hirsutum leaves observed in three phenological stages (vegetative, reproductive, and maturation). The interaction of hyphae with the smooth surface glandular trichomes (a and c), presence of hyphae and fungal reproductive structures interacting with the base of the trichome (b), and ornamented surface glandular trichome covered by hyphae (d)

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Discussion

Cotton plants exhibit low photosynthetic pigment levels, low photosynthetic rates, and photochemical stress in the maturation stage

The differences in the physiological parameters observed for the plants at the maturation and other stages can be explained by the differential reallocation of biomass to the fruits, which is more intense when compared to its reallocation to the reproductive and vegetative parts (Oosterhuis, 1990). During early vegetative growth, most carbohydrates produced by cotton plant leaves are delivered to the root system. Later, however, most carbohydrates are transported to the developing bolls, and the rate of root and aerial growth decreases (Ritchie et al., 2007). This reallocation may increase the plant’s susceptibility during the cotton maturation phase. At this stage, new leaf development decreases while the fruit load increases with the carbohydrate demand, to facilitate fruit development with an aging leaf population (Ritchie et al., 2007). It is estimated that a cotton leaf reaches its maximum photosynthetic capacity at approximately 20 days of age, after which a natural decrease in this capacity is observed. Thus, cotton advances in reproductive growth with the support of an aging leaf canopy, and the physiological and photochemical responses observed in this phase are considered typical of the crop development.

Photosynthesis and photochemistry in the maturation stage of plants infected by F. equiseti are affected more than that during natural aging

Studies have shown that the development of the bolls in the maturation stage is closely related to factors, such as solar radiation and N availability (Li et al., 2009). However, the presence of stimuli stressful to the cotton plant, such as water and saline stress, can reduce the photosynthetic capacity of the crop (Pan et al., 2019; Zahid et al., 2021; Zonta et al., 2017), affecting the homeostasis necessary to ensure productivity. Cases of biotic stress, in disease situations, can affect photosynthetic efficiency, measured by A (net photosynthesis rate) water use efficiency and WUE (water use efficiency), and also photochemical efficiency, measured by ABS/RC (specific light absorption flux per reaction center) and DI0/RC (specific energy dissipation flux at the level of the chlorophylls of the antenna complex) (e.g., Macioszek et al., 2019; Chhabra et al., 2019). In the present study, biotic stress induced by F. equiseti in root-inoculated plants resulted in the lowest A and WUE values and the highest ABS/RC and DI0/RC values. Biotic stress traits observed in fungi belonging to the Fusarium genera are related to the development of the fungus in the conducting vessels, changing root functions (Li et al., 2017). The presence of hyphae hinders water uptake and causes water deficit symptoms (Bhandari et al., 2020) which affects the protoplasm and directly affects photosynthesis. Fusarium-induced water deficit symptoms affect RuBisCo activity and compromise the electron transport rate (Perdomo et al., 2017). Consequently, the presence of these fungi in the roots reduces the uptake of nutrients with water. Chu et al. (2018) explained that this nutritional deficit can reduce the photosynthesis rate, since nutrients act in leaf structural regulation and electron and CO2 transport and can interfere with Calvin cycle processes (Santos et al., 2013; Viégas et al., 2018). Therefore, nutritional restriction associated with the Fusarium-induced water deficit affects the photosynthetic apparatus via photodestruction and photoinhibition (Uarrota et al., 2018).

Root-inoculated cotton plants exhibited high transpiration rates and photochemical stress

The presence of F. equiseti in the roots of cotton plants increases the transpiration rate. In fusariosis events, the increase in E can be dramatic because it amplifies the likelihood of wilting of the plant. In these cases, the plant opens its stomata to ensure CO2 intake, but in return loses a large amount of water. A fine, morphological interaction between the fungi and the cells adjacent to the stomata during infection was verified, and this interaction may affect these structures. As a result, they may lead more effectively toward water loss than carbon uptake, which reflects negatively on the WUE, as this parameter is defined as the amount of carbon assimilated as biomass produced per unit of water used by the crop (Hatfield & Dold, 2019). Conversely, in root-infected plants, the gs values were also high and, when compared to E, indicated the occurrence of uncontrolled water loss unregulated by stomata. Previous studies have confirmed that in Fusarium infected plants, a toxin called fusaric acid secreted by the fungus, can promote disturbances in the leaf temperature, thereby resulting in uncontrolled water loss in the leaves as well as electrolyte leakage due to cell membrane damage (Dong et al., 2012). Fusaric acid causes early hyperpolarization of the root membrane electrical potential which acidifies the extracellular medium and increases membrane permeability (Kuzniak et al., 2001; Gapillout et al., 1996). In general, cellular damage caused by Fusarium infection increases non-stomatal water loss (Wang et al., 2015). This may explain the high E rates observed in inoculated plants. Moreover, since leaf trichomes can also act as transpiratory pathways (Abdulrahaman and Oladele, 2011; Lauter & Munns, 1986), the trichomes of G. hirsutum colonized by fungal hyphae may exacerbate leaf transpiration. Furthermore, cotton plants keep their stomata open during F. equiseti colonization. Previous studies have shown that resistant plants tend to open stomata via defense signaling to promote water loss. Stomatal opening deprives pathogens of water and nutrients, thus, restricting their multiplication (Wu & Liu, 2022).

Biotic stress induced by the root colonization of cotton by F. equiseti also affected the photochemical efficiency, thereby exacerbating the stress parameters. The high ABS/RC ratio indicated that a lower amount of photons was harnessed by the reaction centers than that in PSII. This is consistent with Urban et al. (2017), who explained that the inactivation of reaction centers or the change in antenna size compensate for the dissipation of light energy into thermal energy, resulting in increased DI0/RC values. This reduction in the amount of active reaction centers is considered to be a leaf mechanism to avoid damage in the face of excess light energy absorption (Kalaji et al., 2018).

Non-inoculated plants produced more photosynthetic pigments, had a lower gs, a higher WUE, and higher photochemical efficiency

Indeed, the effects of Fusarium infection at the level of nutrient and water transport negatively impact the photosynthetic system of cotton plants (Gonzales-Soto et al., 2022). Regardless of the type of inoculation, the control plants accumulated more photosynthetic pigments and exhibited high PIABS values. This relationship was observed because chlorophyll a is the main pigment for measuring the changes in fluorescence, given that it is important in dissipating energy from the reaction centers of PSII. In contrast, chlorophyll b participates in PSI as an accessory pigment that helps protect PSII, together with the presence of carotenoids (Dautermann & Lohr, 2017), which help expand the wavelengths absorbed by chlorophyll.

The photochemical imbalance can directly interfere with the synthesis of photosynthetic pigments and lead to the photodestruction of these pigments, consequently compromising the functional integrity of the PSII reaction centers and unbalancing carbon fixation. Biotic stresses can generate diverse symptoms that compromise everything from light energy absorption to anatomical structures. Baghbani et al. (2019) showed that pathogens of the Fusarium genus can actually affect the electron transport sequence, affecting the synthesis and maintenance of chlorophyll (Bassanezi et al., 2002).

The PIABS value, being a more sensitive index, detects and quantifies damage more accurately, since it is possible to relate them to the efficiency of PSII, thereby providing greater accuracy in the correlation between the stresses and electron transport (Sampaio et al., 2012). Thus, the presence of Fusarium hyphae interacting with the cotton plants through the leaves or roots leads to photochemical damage and reduces the PIABS values. High PIABS values indicate high vitality following the increase in the number of chlorophyll reaction centers. This parameter reflects the adequate functions of PSI and PSII (Strasser et al., 2004) which provides information on the synthesis of pigments and photosynthetic performance. It is expected that the inoculation of plants by phytopathogens generates low PIABS and high DI0/RC values, reflecting the harmful metabolic changes in the inoculated plants because they subsequently become dissipation centers to avoid photo-oxidative damage (Roach and Krieger-Liszkay, 2019). In contrast, the lower gs values observed in non-inoculated plants is also an indicator of vitality and the absence of damage, indicating that these plants efficiently regulate water loss through stomata, resulting in a higher WUE (Bertolino et al., 2019).

F. equiseti uses the cells adjacent to the stomata and the base of glandular trichomes to interact with the leaf tissue of G. hirsutum

Being an opportunistic pathogen, the presence of F. equiseti in G. hirsutum tissues depends on the inactivation of some of their defense components as well as the production of metabolites that affect the tissues and make the plant susceptible to infection (Horbach et al., 2011; Oh et al., 2006). One of the infection strategies used by F. equiseti to access the internal leaf epidermal tissue of cotton plants is the penetration of hyphae in the region of contact between the dorsal wall of the stomata and adjacent epidermal cells. According to Pacheco et al. (2021), the ventral wall of the stomata which surrounds the stomatal pore, is thicker and may reach 5 μm depending on the plant species, whereas the dorsal wall is thinner. There is strong evidence of the differential thickening of the guard cell walls in different plants with the outer, inner, and ventral walls being thicker than the dorsal wall (Merced & Renzaglia, 2013, 2014). In typical epidermal cells, the wall reaches a maximum thickness of 2 μm, and because the organization of the wall components in the guard cells (cellulose, xyloglucan, and pectin) is different from that of the epidermal cells (see Rui et al., 2018), it is possible that the contact region may become more fragile. Furthermore, it is known that increased turgescence pressure in guard cells forces their thin dorsal walls to elongate and project into subsidiary cells (Pautov et al., 2017). It is possible that the continuous mechanics of the dorsal wall may also weaken the region in conjunction with the adjacent epidermal cells, therefore, be more amenable to penetration by fungal hyphae. This may be why F. equiseti use this region to penetrate the epidermis of G. hirsutum.

Another region of the adaxial epidermis used by F. equiseti as a gateway to the internal tissues of the leaf is the basal region of the trichomes. Both non-glandular and glandular trichomes are found in the hairy leaves of cotton. The latter are composed of a small peduncular cell and multicellular head and may be globose or have ornamented surfaces (Bondada et al., 2000). These trichomes occupy a special area as a first line of defense and management against sucking insects and pests (Ahmed et al., 2020). These structures produce pest- or pollinator-interacting phytochemicals that are stored or volatilized on the plant surface, and are specific sites for the biosynthesis and excretion of secondary metabolites (Venditti et al., 2014). Wang et al. (2021) showed that the cells of the glandular trichomes of Rosa roxburghii have thin cell walls and frequent plasmodesmata with large intercellular spaces. The outer wall of this type of trichome appears even thinner in the basal region, which was observed by Argyropoulou et al. (2010) when analyzing the secretory trichomes of Lippia citriodora. This may help to explain the use of this basal region for the penetration of F. equiseti hyphae into the epidermal tissue of G. hirsutum.

Studies have shown that G. hirsutum trichomes synthesize gossypol and related compounds, such as dimeric di-sesquiterpenes which are natural pesticides (Dayan & Duke, 2003) and exhibit antifungal activity (Mellon et al., 2012). Despite the metabolites secreted by the glandular trichomes of the adaxial surface of cotton leaves, F. equiseti colonizes these structures, showing resistance and insensitivity to these active compounds. Conversely, the fragmentation of the terminal regions of the hyphae suggests that this fragmentation aims to increase the contact area of the phytopathogen with the plant tissue. Thus, in these contact regions, Fusarium secretes toxins that affect the host and increase the colonization capacity of the fungus. Several virulence factors have been reported from F. oxysporum. These factors play important roles at different stages of the infection process to induce disease and neutralize plant defences (Roncero et al., 2003). The most studied virulence factor of Fusarium is fusaric acid which participates in pathogenicity by reducing plant cell viability (Bani et al., 2014). In addition, some F. oxysporum isolates produce additional toxins, such as beauvericin, enniatin B, bikaverin, moniliformin, fumonisin, and trichothecenes (Moretti et al., 2022; Irzykowska et al., 2012; Son et al., 2008) which also contribute to the pathogenicity. Recently, Almasoodi, and Naser (2022) observed a strong correlation between the pathogenicity and toxins produced by F. solani, such as deoxynivalenol and T2 toxin. There have been few reports on the mycotoxins produced by F. equiseti, but Jurado et al. (2005) showed that F. equiseti, F. sporotrichioides, and F. poae can produce trichothecene type A, and are the major trichothecene-producing Fusarium species. These species are common cereal fungal pathogens and cause Fusarium head blight in small-grain cereals and ear rot in maize.

Thus, even though it is considered an opportunistic pathogen for G. hirsutum, F. equiseti can colonize the plant tissues during different phenological stages, thus, affecting the production of photosynthetic pigments, gas exchange parameters, and photochemical efficiency, which can be predicted to culminate in losses in crop productivity.

Conclusions

Cotton plants incur metabolic losses in the maturation stage, resulting in low photosynthetic pigment levels in leaf tissues, low photosynthetic rates, and photochemical stress. However, when infected by F. equiseti in the root region, the photosynthesis and photochemistry of these plants are further affected, as evidenced by the low A and WUE rates and high ABS/RC and DI0 /RC values. Notably, the presence of F. equiseti in the roots of cotton plants, regardless of the phenological stage evaluated, results in high transpiratory rates, suggesting non-stomatic water loss. This is possibly exacerbated by fungal colonization of the leaf trichomes and photochemical damage, and is also characterized by high ABS/RC and DI0/RC values. In contrast, the non-inoculated (control) plants produced more photosynthetic pigments and exhibited higher WUE and photochemical efficiency values, as evidenced by the high PIABS values. The evaluation of the leaf histopathology of the plant-phytopathogen interaction showed that more fragile regions of the epidermis, such as the contact region between guard cells and adjacent epidermal cells, as well as the basal region of glandular trichomes, occurred at all developmental stages constituting important access routes for F. equiseti to the inner leaf layers. Alternatively, hyphae, followed by thinner hyphae, may constitute a strategy to facilitate colonization. This study is important for not only highlighting the physiological basis of the susceptibility of cotton plants to F. equiseti at different developmental stages, but also helping to elucidate the mechanisms used by Fusarium species to interact with leaf tissue during pathogenesis.