Investigating Biochemical and Histopathological Responses between Raspberries and Aculeastrum americanum

Late leaf rust is a fungal disease in raspberries caused by Aculeastrum americanum (Farl.) M. Scholler U. Braun (syn. Thekopsora americana (Farl.) Aime McTaggart) leading to early defoliation and yield losses. Red raspberries (Rubus idaeus L.) are susceptible to this pathogen, although this susceptibility varies among cultivars. In contrast, black raspberries were previously reported to be more resistant (Rubus occidentalis L.) and immune (Rubus niveus Thunb.) to this pathogen, raising their importance in plant breeding programs. However, what features make them respond differently to the same pathogen? In this study, we characterize for the first time the pre- and post-formed structural and biochemical defense mechanisms of R. idaeus cv. Autumn Bliss, R. occidentalis and R. niveus. Ultrastructural and histopathological analyses were used to uncover the interactions between these raspberries and A. americanum. The ultrastructural results indicate that the pathogen germinates on both leaf surfaces but can only form appressoria on the stomata. Although the three raspberry species were infected and colonized by A. americanum, a clear difference in susceptibility was observed between them. A compact mesophyll, pre- and post-formed phenolic compounds, and post-formed pectic compounds were the main plant defense mechanisms against fungal colonization. These findings provide new information about raspberries’ defense mechanisms in response to A. americanum and elucidate the interactions occurring in these pathosystems.

The main symptoms are powdery yellow spots, which correspond to reproductive structures called uredinia and are found in all aerial parts of the infected plants [1,9]. Symptomatic fruits become unfit for sale, and leaves may drop prematurely, causing severe yield losses [1,10,11]. These are some outcomes of plant tissue colonization that lead to structural and physiological responses during the plant-pathogen interaction. However, it is still unknown which defense mechanisms raspberries deploy against the colonization process of A. americanum.

Plant Material
Raspberry plants of R. idaeus cv. Autumn Bliss, R. occidentalis and R. niveus were grown in 7 L pots with a commercial peat substrate (Agrolink Biogrow, VDK) and kept in a greenhouse. The water was supplied by drip irrigation, and slow-release fertilizer was added every eight months at 5 g L -1 (N:P:K 15-09-12, Osmocote Plus ® , Bloomington, IN, USA).

Light Microscopy (LM): Qualitative and Quantitative Analyses
Five healthy fully expanded leaves per species collected from different plants were sampled for qualitative and quantitative analysis of the anatomical structure. Samples were collected from the middle third of the terminal leaflet to maintain standardization (n = 5). The following anatomical traits were measured: total leaf thickness, cuticle thickness, the height of the epidermal cells on both leaflet surfaces, total mesophyll thickness, thickness of the palisade and spongy parenchyma, and the area of the intercellular space. Three measurements were conducted on five cross-sections per leaflet to obtain the average for each trait in each raspberry species.
For determining the anatomical structure, 1 cm 2 fragments of each sample were fixed for 48 h in formalin-ferrous sulfate for detection of phenolics [36] or in Karnovsky's solution [37], subjected to a vacuum pump to remove the air from the intercellular spaces, and dehydrated in an increasing ethyl series up to 100% ethanol and infiltrated in hydroxyethyl-methacrylate (Leica Historesin, Heraeus Kulzer, Hanau, Germany). The obtained blocks were sectioned in a rotary microtome (Leica RM2245, Leica Biosystems, Heidelberg, Germany) at 5 µm thickness. Cross-sections from samples fixed in Karnovsky's solution were stained with 0.05% toluidine blue (pH 4.5) [38] and mounted in water for structural analyses, or stained with Sudan IV [39] to detect lipids to delimit the cuticle. Cross-sections fixed in formalin-ferrous sulfate were mounted on slides in water for detection of the phenolic compounds.
To analyze the stomatal index (SI) and the morphometry of the stomata, five terminal leaflets were oven-dried at 37 • C for 20 min, then 1 cm 2 of the abaxial surface in the middle third of the leaflet was shaved under a stereomicroscope using sharp bent tweezers to remove the non-glandular trichomes. These samples were submerged in a 5% (w/v) sodium hydroxide aqueous solution for 30 min, rinsed in distilled water and placed in a 20% (v/v) sodium hypochlorite solution until complete decolorization. The fragments were rinsed in distilled water, stained with Alcian Blue-Safranin O (9:1 v/v) [40], mounted in 50% glycerol gelatin and analyzed under the microscope. The SI was determined by counting all the stomata (S) and epidermal cells (E) in the microscopic view field and applying the formula (SI% = S/(S + E)) × 100 [41]. Morphometry of the stomata was performed by measuring the length and width of each stoma in the microscopic view field.
For quantification of the crystal idioblasts, 1 cm 2 fragments sampled from the middle third of five leaflets were decolorized overnight in an ethanol/acetic acid solution (6:1 v/v) [42], rinsed in distilled water and mounted in 50% glycerol gelatin. The crystal idioblasts were visualized under polarized light, and the images were captured at 400× magnification. All crystals observed in the selected images were counted regardless of their size.
The images for qualitative and quantitative analysis were documented with a Leica DMLB microscope (Leica Microsystems) coupled to a digital camera (Leica DFC 310Fx). All measurements were performed in Image J Software version 1.52t [43].

Obtention and Maintenance of Aculeastrum americanum Inoculum
The monopustular isolate of Aculeastrum americanum was genetically characterized by Ribeiro and Spósito (GenBank: MW039448 corresponds to the ITS and MW039395 corresponds to the small subunit ribosomal RNA gene 18S) and inoculated in R. idaeus cv. Heritage for maintenance [44]. The isolate was collected in a commercial orchard in the municipality of São Bento do Sapucaí, São Paulo, Brazil (22 • 37 34.5" S 45 • 34 50.4" W) [27]. In every inoculation trial the viability of urediniospores was verified by a germination test in which three drops of the spore suspension were deposited in three Petri dishes and incubated for 24 h in a dark and humid chamber. Then, we added lactoglycerol to each drop and counted the germinated spores in 100 spores per drop.
In all experiments, 2-week-old spores were used to prepare a suspension at the concentration of 10 5 urediniospores mL −1 in distilled water and Tween 20 (0.01%), and the spores' viability was evaluated [44]. The leaves were inoculated by spraying the suspension on the entire leaf blade or by depositing drops (50 µL) on delimited areas. Leaves sprayed with distilled water were considered as the control. After inoculation, the plants were kept at 24 • C in a dark and humid chamber for 24 h, and then all plants were placed in a greenhouse until sample collection. Terminal leaflets of fully expanded leaves collected from four plants of each raspberry species were used for the surface analysis. The inoculations were performed by adding spore suspension drops or distilled water to a delimited area on the leaflets' abaxial or adaxial surface. The samples were collected 6, 12, 24, 36 h and 14 days after inoculation from three leaves per period. This experiment was performed three times. Inoculated and healthy samples were fixed in Karnovsky's solution [37] for 48 h and subsequently dehydrated in an ethanolic series [19]. Next, they were critical-point-dried [45] (Baltec EM CPD 300), mounted on aluminum stubs and coated with a thin layer of gold (30-40 nm) by a sputter coater (Balzers, model SCD 050). The analyses and electron micrographs were performed using a scanning electron microscope (model JEOL JSM-IT300LV) operated at 15-20 kV. The images were coloured using Adobe Photoshop 2020.

Light Microscopy (LM)
Fully expanded leaves from the three raspberry species were inoculated on the abaxial surface with an A. americanum spore solution or distilled water as described in Section 4. The samples were collected 14 days after inoculation (dai), and the lesions were immediately documented using a Leica DFC295 camera coupled to a Leica M205 C stereoscopy microscope. Afterwards, fragments of the samples were fixed in Karnovsky's solution [37] for 48 h, dehydrated and infiltrated in hydroxyethyl methacrylate [19]. The samples were transversally sectioned to 5-7 µm using the rotary microtome described previously. The sections were stained with 0.05% toluidine blue (pH 4.5) [38] and mounted on slides in water. When required, other slides were stained with ruthenium red to confirm the presence of pectin and acidic mucilage [36]. Images were captured as described in Section 2.3.

Statistical Analyses
The statistical significance of the biochemical and anatomical traits was determined by one-way ANOVA followed by Tukey's post-hoc test (p < 0.05) using RStudio software 2018 version 1.2.5033 [46].

Characterization of Pre-Formed Defense Mechanisms in Raspberries
The pre-formed biochemical compounds and structural defense mechanisms were investigated in red and black raspberries. The leaves showed variation in their morphology, although all three species presented compound leaves with toothed margins (Figure 1a sections were stained with 0.05% toluidine blue (pH 4.5) [38] and mounted on slides in water. When required, other slides were stained with ruthenium red to confirm the presence of pectin and acidic mucilage [36]. Images were captured as described in Section 2.3.

Statistical Analyses
The statistical significance of the biochemical and anatomical traits was determined by one-way ANOVA followed by Tukey's post-hoc test (p < 0.05) using RStudio software 2018 version 1.2.5033 [46].

Characterization of Pre-Formed Defense Mechanisms in Raspberries
The pre-formed biochemical compounds and structural defense mechanisms were investigated in red and black raspberries. The leaves showed variation in their morphology, although all three species presented compound leaves with toothed margins (   The cross-sections of the terminal leaflets showed a uniseriate epidermis on both surfaces, with larger cells on the adaxial face (Figure 1d-f). The cuticle thickness was higher in R. niveus on the adaxial surface than in the other two species (Table 1). The epidermal cells were smaller in R. niveus than in the other species on both leaf surfaces (Table 1, Figure 1f). The three raspberries were hypostomatic with anomocytic stomata slightly positioned above the level of the epidermis (Figure 1g-i). The width and length of the stomata were smaller in R. niveus than in R. idaeus and R. occidentalis, which presented similar values (Table 1). Although numerous stomata were observed in all three species, the stomatal index was higher for R. niveus, which was statistically different from R. idaeus (Table 1). In all studied species, both epidermises showed trichomes, which were more frequent on the abaxial side. The mesophyll was dorsiventral, and its thickness did not differ among the species (Figure 1d-f, Table 1). It was more compact in R. niveus because of the observed two layers of palisade parenchyma, the lower spongy parenchyma thickness and almost no intercellular space (Figure 1f, Table 1). In general, R. idaeus and R. occidentalis had one layer of palisade parenchyma (Figure 1d-e), while R. occidentalis had a greater intercellular space than the other two species (Table 1). Idioblasts containing crystals of the druse type were frequently found in all species along the mesophyll (Figure 1d) and the midrib. Interestingly, R. occidentalis presented fewer druses than the other two raspberries (Table 1). Phenolic compounds were detected in the epidermal, mesophyll and vascular bundle cells in all species. However, the reaction intensity was lower in R. idaeus (Figure 1j) than in black raspberries (Figure 1k-l).
The observation (Figure 1j-l) that black raspberries had more constitutive phenolic compounds than R. idaeus was confirmed by quantitative analyses. Indeed, R. occidentalis and R. niveus presented twice as many total phenolics than R. idaeus (Table 1). Likewise, the proanthocyanidins stood out in these species, with higher values for R. occidentalis (1.20 ± 0.02) and R. niveus (0.87 ± 0.02). Once more, the antioxidant capacity determined by the DPPH method showed higher percentages for black raspberries (Table 1). Interestingly, the total flavonoid content was three times lower in R. occidentalis and R. idaeus compared with R. niveus ( Table 1). The chlorophylls a, b and, consequently, the total chlorophyll, were significantly higher for R. niveus and were not different between R. occidentalis and R. idaeus.
The same was observed for total carotenoids (Table 1). Overall, black raspberries presented the highest values for the studied biochemical traits.

Ultrastructural and Histopathology of the Interaction between Raspberries and Aculeastrum americanum
Scanning electron microscopy analysis showed the first interactions between the raspberry species and Aculeastrum americanum. The pathogen viability evaluated in vitro had a range of 38% to 65% germination. The urediniospores inoculated on the abaxial leaf surfaces and observed at 6 h after inoculation (hai) germinated and formed a single (Figure 2a) or multiple germ tubes (Figure 2b). Although the germ tubes could reach lengths greater than 100 µm, they had a medium to long elongation (10-100 µm) overall. It was noted that the germ tube branched in different directions when in contact with the epidermis (Figure 2c,g). Despite the urediniospore germination that occurred in R. niveus (Figure 2d), the formation of an appressorium was observed only at 12 and 36 hai in R. occidentalis and R. idaeus (Figure 2e,f).
To confirm that penetration occurred only via the stomata, the leaves' adaxial surfaces were inoculated with A. americanum urediniospores and analyzed at 24 hai. Similar to the abaxial surface, the spores germinated, forming elongated and branched germ tubes, but no appressorium was observed (Figure 2g). The late leaf rust latency period was 7-10 days for all plants, when satellite uredinia (Figure 2h-i) emerged on the leaves' abaxial surface with the same morphological features in all the raspberry species.
Mesophyll colonization in all the studied species was strictly intercellular, and the hyphae were found mainly in the spongy parenchyma (Figure 3a-c), especially in the substomatal chamber (Figure 3a,b). The pathogen grew intracellularly, forming the haustorium, consisting of the neck and haustorial body (Figure 3d,e). The mesophyll cells of all raspberries accumulated more phenols as a response to fungal colonization (Figure 3a-c,f), but particularly the black raspberries, which also showed the presence of pectic materials (Figure 3f,h,i) compared with uninfected areas (Figure 3g). The formation of uredinia began mainly in the region of the substomatal chamber (Figure 3j).
The visible symptoms observed on the adaxial surfaces (Figure 4a,e,i) 14 days after inoculation (dai) with A. americanum corresponded to the yellowish uredinia on the abaxial surfaces (Figure 4b,f,j). R. idaeus had a chlorotic lesion on the adaxial surface (Figure 4a) without apparent necrosis. In contrast, this necrotic characteristic was distinguishable in R. occidentalis and R. niveus (Figure 4e,i), since the pustules emerged on the abaxial surface. In R. idaeus, the necroses on the adaxial surface were identified many days afterwards. Interestingly, the pustules emerging in black raspberry leaves were limited by the leaves' venation areoles (Figure 4f,g,j,k), in contrast to the outspread pustules found in R. idaeus (Figure 4b,c). During the process of image capture, the pustules sporulated, releasing the urediniospores observed outside the area surrounded by the veins (Figure 4j). The mesophyll cells of R. occidentalis (Figure 4g,h) and R. niveus (Figure 4k,l) were intensely stained blue/greenish-blue by toluidine blue because of the higher content of phenolic compounds than R. idaeus (Figure 4c,d).  hyphae were found mainly in the spongy parenchyma (Figure 3a-c), especially in the substomatal chamber (Figure 3a,b). The pathogen grew intracellularly, forming the haustorium, consisting of the neck and haustorial body (Figure 3d,e). The mesophyll cells of all raspberries accumulated more phenols as a response to fungal colonization (Figure 3ac,f), but particularly the black raspberries, which also showed the presence of pectic materials (Figure 3f,h,i) compared with uninfected areas (Figure 3g). The formation of uredinia began mainly in the region of the substomatal chamber (Figure 3j).  wards. Interestingly, the pustules emerging in black raspberry leaves were limited by the leaves' venation areoles (Figure 4f,g,j,k), in contrast to the outspread pustules found in R. idaeus (Figure 4b,c). During the process of image capture, the pustules sporulated, releasing the urediniospores observed outside the area surrounded by the veins (Figure 4j). The mesophyll cells of R. occidentalis (Figure 4g,h) and R. niveus (Figure 4k,l) were intensely stained blue/greenish-blue by toluidine blue because of the higher content of phenolic compounds than R. idaeus (Figure 4c,d).

Discussion
Structural and biochemical mechanisms have been demonstrated to help plants manage biotic and environmental stresses. This study uncovered some of these mechanisms in three raspberry species in response to the interaction with A. americanum, the causal agent of late leaf rust.
Previous anatomical studies on leaves from different raspberry species and varieties indicate that they are similar in most aspects, with details in some structures such as the epidermal cells and covering trichomes [47][48][49]. Because our focus was on analyzing physical barriers as potential defense mechanisms against A. americanum, the morphology and histochemistry of the different types of trichomes found on black raspberries could still be further investigated in the future. Even so, for all three raspberries, our results confirm the presence of very long unbranched non-glandular trichomes that are strongly tangled. According to Karley et al. [50], these trichomes negatively affect herbivore-R. idaeus interactions. In contrast, Wang et al. [51] suggest that the high density of trichomes contributes to the adhesion of fungal spores and produces a microclimate that favors spore germination. Indeed, even covering the raspberry leaf surface, the non-glandular trichomes are not a fully effective barrier against A. americanum.
The height of the cuticle coating the entire epidermis, including the stoma guard cells, was measured to check its potential role as a pre-formed resistance barrier against A. americanum. However, no evidence of resistance to the pathogen was found on any of the raspberry leaves. In fact, the cuticle's role is more commonly discussed in terms of interactions in which the pathogen penetrates directly into the host [52,53].
It has been previously described that hypostomatic leaves have anomocytic stomata located above the epidermis level for R. idaeus varieties and R. loganobaccus [47,48,54]. Herein, we confirm these characteristics for R. idaeus cv. Autumn Bliss and, for the first time, document them for R. occidentalis and R. niveus. Among the studied raspberries, R. niveus has stomata with a smaller width and length, and a higher stomatal index. Having more stomata available should apparently facilitate the pathogen's penetration [55][56][57], however, nothing that drew attention was observed for this black raspberry. Nevertheless, the morphology of the stomata is also related to the penetration process, as a small stoma might hinder the formation of an appressorium [58]. Studies addressing the composition of the wax covering the stoma and the relationship between disease severity and the stomatal index might help to uncover the role of the stomata in the A. americanum-raspberry pathosystem.
In all studied species, the urediniospores germinated on both leaf surfaces, yet only formed an appressorium above the stomata on the abaxial face. This strategy has been commonly observed for several rust fungi [59][60][61], although the appressorium can also be formed over epidermal cell junctions in other rust pathosystems [22,62,63]. Indeed, the medium to long germ tubes observed for A. americanum are a typical characteristic of urediniospores that penetrate the host through the stomata [64,65]. Moreover, the multiple germ tubes emitted by A. americanum may favor infection processes and increase the aggressiveness of the pathogen [66]. Another characteristic observed was the ability of the germ tube to branch. Although this event may be influenced by external conditions, such as the time of incubation and the temperature during germination [67], ramification of the germ tube was common in all experiments performed using the optimal temperature for A. americanum.
Following penetration, the hyphae of A. americanum developed in the intercellular spaces of the mesophyll, mainly in the substomatal chamber and alongside the spaces of the spongy parenchyma. The hyphae were hardly seen in the palisade parenchyma, probably because its cells are more columnar and stacked side by side, which may hamper the development and visualization of hyphae. Moreover, R. niveus had a compact double-layered palisade parenchyma and presented the lowest intercellular values, traits that act as physical barriers to the growth of hyphae. It has been found that thicker palisade tissue exhibits greater resistance to pathogens such as Puccinia zoysiae [58] and Xanthomonas arboricola [68].
Meanwhile, R. occidentalis had more intercellular spaces among the studied raspberries, presumably allowing for the development of hyphae. Interestingly, the constitutive and post-infection biochemical traits of black raspberries seem to be more effective against A. americanum. The intercellular spaces of infected leaves of R. occidentalis and R. niveus were filled with pectic substances, in contrast to those of R. idaeus. Likewise, grapevine leaves infected by Phakopsora meliosmae-myrianthae (syn. P. euvitis) deposited pectin on the cell wall and intercellular spaces [69]. Resistant genotypes of Coffea spp. inoculated with the rust Hemileia vastatrix also accumulated pectin and polysaccharides in the intercellular spaces, which was not observed in healthy and susceptible tissues [70]. It is clear from these findings that pectins are important defense mechanisms against plant pathogens, which merits further investigation.
Haustoria were frequently observed inside R. idaeus cells, while black raspberries accumulated more phenolic compounds after colonization by the fungus, which may have hindered their observation. In general, the mature haustorium was about 10 µm in length and had a cylindrical to allantoid shape, as described by [71].
The biochemical and histochemical results show that black raspberries have higher levels of constitutive phenolic compounds than R. idaeus, which might explain the more effective response to colonization by A. americanum in R. occidentalis and R. niveus.
Phenolics include several classes of compounds that are known for their antimicrobial activity [72,73]. Similar to phenolic compounds, black raspberries displayed higher antioxidant activity, as assessed by the DPPH method. These results corroborate the significant correlation between phenolic substances and antioxidant activity found by Oszmiański et al. [74] in Rubus leaves. Surprisingly, the flavonoid content was lower in R. occidentalis. However, it might be compensated by different phenolics such as proanthocyanidins and others not explored in this study but found in the Rubus species [75][76][77]. Additionally, flavonoids, proanthocyanidins and carotenoids act as antioxidants to scavenge free radicals produced during plant-pathogen interactions [78]. The staining with DAB (3,3 -diaminobenzidine) for the detection of hydrogen peroxide and with NBT (nitroblue tetrazolium) for the detection of superoxide anion did not produce any results in all raspberry species studied, even when adapting the protocols. We attributed this failure to their dense leaf pubescence promoting a nearly impenetrable layer on the abaxial leaf surface [49,75,79,80].
The epidermal and mesophyll cells of infected R. occidentalis and R. niveus accumulated polyphenols that may vastly hamper the development of haustoria. The accumulation of phenolic compounds as a response to pathogen infection has been confirmed for several pathosystems [19,20,24,69,81]. Their role is not only associated with the chemical activity but also with the structural function. Polyphenols added to the cell wall can strengthen the structure and make the formation of haustoria difficult [81]. Plants also produce lignin-like compounds by activating the phenylpropanoid pathway in response to infection [70,72,82].
It has been shown that the vascular bundle restricts pathogen colonization in Vitis vinifera cultivars [69,83]. Interestingly, it was only on the leaves of black raspberries that the uredinia appeared to have been delimited by the veins 14 days after inoculation with A. americanum. Nevertheless, as the infection progressed and more nutrients were needed to form uredinia, colonization also spread beyond the vascular tissues.
In conclusion, a more compact mesophyll, pre-and post-formed phenolic compounds, and post-formed pectic compounds are the main defense mechanisms found in raspberries that play a role against A. americanum. Although the raspberries had both pre-formed and post-formed defense mechanisms, these were not sufficient to totally contain infection and colonization by A. americanum. According to these results, we confirm the susceptibility of R. idaeus cv. Autumn Bliss to late leaf rust and show the absence of immunity to A. americanum for R. occidentalis and R. niveus.