Influence of the Phytosanitary Status, Cultivar, and Harvest Time on the Phenolic, Chlorophyll, and Alkaloid Content of Rosa sp. Leaves

Abstract

Diplocarpon rosae Wolf is the most common and damaging fungal pathogen in roses. Nationwide, the attack of this pathogenic fungus is very serious in most rose gardens due to rapid disease development that typically leads to leaf yellowing and defoliation. This study aimed to assess the way in which the fungus’s attack affects the chemical composition of Rosa sp. leaves. The research was conducted in the summer of 2023 on ten cultivars of rose grown in the rosary in the ‘Alexandru Buia’ Botanical Garden in Craiova. The influence of the cultivar and harvest time was discussed. Leaf black spot on roses produced by the fungus Diplocarpon rosae Wolf affected plants in all cultivars and the differences between cultivars highlighted the sensitivity of the relationship between plants and pathogens when exposed to some microclimatic environment factors. The correlations of attack degree with tannins, chlorophyll, or alkaloids were significant, negative for tannins (r = −0.189 *) and chlorophyll (r = −0.517 ***) and positive for alkaloids (r = 0.510 ***). Between phenolic compounds, tannins, flavonoids, and chlorophyll, very significant positive correlations were found. Alkaloids established negative and also very significant correlations with phenolic compounds (r = −0.403 ***), tannins (r = −0.339 ***), flavonoids (r = −0.409 ***), and chlorophyll (r = −0.604 ***).

1. Introduction

The rose (Rosa sp.) belongs to the Rosaceae family. It is an important flowering plant used both in the cosmetics industry and for ornamental purposes due to its fragrance and appearance [1]. The diversity of its morphological characteristics, such as color, fragrance, and size, but also its adaptability to the environment and the long flowering period have contributed to its intensive spread and cultivation [2].

The present study aimed to assess the impact of Diplocarpon rosae Wolf’s attack on the chemical composition of rose leaves. Polyphenols are among the most important bioactive compounds in a plant due to their anti-inflammatory, antioxidant, anti-proliferative, antimicrobial effects, and enzymatic implications [3]. The phenolic composition can be influenced by several factors, for example: the period and place of collection, storage conditions, processing (time, temperature, pressure, and solvents), etc. [1,3,4]. Therefore, qualitative and/or quantitative determination of polyphenolic compounds in plants is necessary. Numerous research projects on rose biology have shown that the resistance of wild rose to pathogens, such as Diplocarpon rosae Wolf, is much higher than in cultivated ones [5]. Other elements that influence resistance to infection are age and variety, the latter being able to also influence the maintenance cost of the plant [2].

Fungal disease is a major source of damage to roses. The main fungi that wreak havoc in the Romanian rose gardens include Diplocarpon rosae Wolf, Alphitomorpha pannosa, and Cercospora puderi. Of these, black spot is considered the most serious disease of roses (Rosa sp.), Diplocarpon rosae Wolf being a hemibiotrophic fungus that infects members of the genus Rosa [6]. Diplocarpon rosae can be found on some species of wild roses and most varieties of roses grown in gardens. It deforms leaves and reduces the vitality of sensitive plants unless they are regularly treated with systemic fungicides [7]. However, as a result of public concerns and increasing legal restrictions on pesticide use in parks and gardens, the availability of effective fungicides is steadily decreasing. Therefore, rose growers are interested in developing attractive roses with a high level of strength. A prerequisite for this is the availability of resistant germplasm as a source of effective resistance genes. Because of limited genetic diversity among rose varieties, most of which are highly susceptible, the search for new resistance genes focuses on wild rose species [8].

Infection of rose plant genotypes susceptible to Diplocarpon rosae Wolf leads to the appearance of black spots on the leaves, surrounded in many cases by chlorotic areas. The damage caused by this disease is not only indirect, due to the loss of the aesthetic value of the commercial product, but also impacts directly by weakening plants to the point of killing highly susceptible genotypes. Propagation of the pathogen occurs mainly by asexual conidia, thus, the disease spreads rapidly through water and direct contact [6,9].

Phenolic substances are toxic to pathogenic microorganisms and increase the physical and mechanical strength of the host cell wall [10]. The oxidation of these phenolic compounds using polyphenol oxidase produces quinones (antimicrobial compounds), which are toxic to invading fungi, thus, providing resistance against a wide range of pathogens [11]. Phenylalanine ammonia-lyase, one of the key enzymes in the phenylpropanoid pathway, has a role in the synthesis of phytoalexin and salicylic acid. Increased chipboard activity subsequently increases the content of phenolics, providing resistance to plant diseases [12].

Understanding the mechanism of plant resistance against pathogens at different levels provides new opportunities for producing improved varieties with better disease resistance. There have not been many studies in this line on infection with black spot disease in roses. Saidulu et al. (2022) [10] investigated the role of different defense-related enzymes and compounds during different periods of progression after infection with the black spot pathogen in different rose genotypes that possess differential resistance. The attack of two pathogens, Sphaerotheca pannosa var. rosae and Diplocarpon rosae Wolf, in some rose varieties revealed changes in plant growth and development [13]. Thus, through the attack of Diplocarpon rosae on the roses, the length of the leaves was reduced between 3.7% (‘Paul McCartney’) and 18.7% (‘Melina’). Their width was reduced between 0.9% (‘Paul McCartney’) and 14.0% (‘Melina’), with even the diameter of the flower suffering reductions between 7.7% (‘Terracotta’) and 14.1% (‘Paul McCartney’). In order for the roses to achieve the purpose for which they are grown, one must know the harmful agents whose attack can lead not only to quantitative deteriorations, but especially to qualitative and sometimes difficult to estimate deteriorations.

Fourier transform infrared spectroscopy (FTIR) is a useful method for early detection of rose disease. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) generates an IR spectrum comprising bands from all cellular components (membranes, proteins, and nucleic acids) and evaluates the entire composition of an organism cell [14,15]. The created spectrum can be used to identify the biochemical or metabolic ‘fingerprint’ of a sample by measuring the vibrations of bonds within chemical functional groups. By obtaining IR spectra from plant samples, one can detect minor changes in primary and secondary metabolites [16,17].

This paper aimed to quantify the attack produced by the fungus Diplocarpon rosae Wolf in some varieties of roses grown in the rosary of the ‘Alexandru Buia’ Botanical Garden in Craiova. The pathogen attack on rose leaves was quantified by frequency, intensity, and degree of attack. FTIR spectroscopy was used to realize a correlation between the spectra of FTIR and the health status of roses. The analysis of the content of some bioactive compounds in the healthy and affected leaves, at three different moments (28–29 June, 12–13 July, and 4–5 September 2023), before and after treating the roses with a mixture of Faster 10 CE, Ditan, and Nissorun 10 WP (conducted on 7 July 2023) aimed at emphasizing the influence of the phytosanitary status, cultivar, and harvest time on the rose leaves.

2. Materials and Methods

2.1. Plant Material and Sampling

The research was conducted in the summer of 2023 on ten varieties of rose (‘Orient Express’, ‘Paul McCartney’, ‘Terracotta’, ‘Glorious’, ‘Mythos’, ‘Holstein Perle’, ‘Melina’, ‘Asja’, ‘Velvet Fragrance’, and ‘Cluj 2010’) grown in the rosary in the ‘Alexandru Buia’ Botanical Garden in Craiova, belonging to the University of Craiova. The university botanical garden is located in the south-west of Craiova municipality (altitude of about 99 m, latitude 44019′24.3″ N, and longitude 23047′17.9″ E), on an area of approximately 12.8 hectares, and the rosary is located in the south-west of the garden, in the nursery sector.

The plants were inspected frequently to determine the presence of some pathogens. The presence of the fungus Diplocarpon rosae Wolf was detected early and the ‘black spot’ disease of roses was carefully monitored. The fungus inoculation was carried out spontaneously without intervening in the pathogenesis process until the necessary treatments were carried out. Simultaneous to the installation and evolution of the disease, no other spontaneous pathologies were noted on the surface of the plant or in the anatomical–physiological structure. The periodic observations followed the determination of the attack frequency, intensity, and degree on each rose cultivar. The ‘black spot’ disease is considered to be one of the most injurious diseases of outdoor grown roses in Romania, causing great damage depending on the climatic conditions each year but also on the phytosanitary program. The control of pathogen propagation in culture heavily relies on fungicides. Thus, all roses from the nursery sector were treated on 7 July 2023 with a mixture of Faster 10 CE, Ditan, and Nissorun 10 WP.

To estimate the pathogen attack on the Rosa sp. cultivars, the mature leaves from the median area of plants were analyzed. The severity of the attack was estimated using the calculation formulae elaborated by Săvescu and Rafailă (1978) [18], using the length and width of sheets related to attacked and unattacked organs. The attack frequency and intensity were obtained as average values of ten determinations. The number was considered sufficient because the errors in this case were very low [19]. Based on the attack degree (AD) values calculated according to the average values of the attack frequency and intensity, the following resistance classes were used to classify the analyzed cultivars [13,20]: resistant (R, with AD = 0–1%), medium resistant (MR, with AD = 1–5%), sensitive (S, with AD = 5–10%), and very sensitive (FS, with AD > 10%).

2.2. Chemicals and Reagents

All reagents used in this research were analytical grade reagents and were obtained from Merck-Sigma-Aldrich, Darmstadt, Germany.

2.3. Extraction Procedures and Biochemical Determinations

For the extraction of polyphenols, flavonoids, alkaloids, and tannins from rose leaves, three ground samples of 1 g leaves in 10 mL methanol (for the first three biochemical compounds) or distilled water (for the extraction of tannins) were used. All samples were subjected to the following protocol: vortexing for 5 min at 3000 rpm, ultrasonication for 30 min at 40 kHz, and centrifugation at 6500 rpm for 30 min [21].

An amount of 1 mL filtered extract was used for the determination of these bioactive compounds, according to the methodology proposed by Giosanu et al. (2018) [22] (for polyphenols), Giura et al. (2019) [23] (for flavonoids and tannins), and Singh et al. (2004) [24] (for alkaloids). The content of polyphenols (TPC) and tannins (TTC) was expressed as mg gallic acid equivalents (GAE) 100 g⁻¹ fresh leaves. Similarly, the flavonoid’s (TFC) and the alkaloid’s content were expressed as mg catechin equivalents (CE) 100 g⁻¹ fresh leaves and mg atropine sulfate equivalents (AE) 100 g⁻¹ fresh leaves.

Determination of chlorophyll content was made according to the methodology proposed by Vijan et al. (2019) [25], using 0.1 g of rose leaves and 10 mL of 90% acetone for extraction. Similarly, the chlorophyll content was expressed as mg 100 g⁻¹ fresh leaves.

2.4. ATR-FTIR Analysis

The spectral measurements were made with an FTIR Jasco 6300 spectrometer. An ATR accessory equipped with a diamond crystal (Pike Technologies, Madison, WI, USA) allows the collection of FTIR spectra directly from a sample without any special preparation. The spectra were recorded in the region of 4000–400 cm⁻¹, detector TGS, apodization Cosine. The spectral data were processed with JASCO Spectra Manager II software, Easton, PA, USA. Samples were scanned at 4 cm⁻¹ resolution, accumulation: 100 scans. In all cases, rose leaves (healthy and diseased) were collected in batches of ten samples. ATR-FTIR spectra were obtained from every tissue of leaves (healthy and diseased). For each group of tissue, an average spectrum was used from the Spectra Manager II software for further analysis.

2.5. Statistical Analysis

Statistical analysis was performed with the trial version of IBM SPSS Statistics 26.0 software package, Armonk, New York, USA. A total of 180 samples of rose leaves (90 healthy and 90 affected by Diplocarpon rosae Wolf’s attack) were analyzed at three different moments (28–29 June, 12–13 July, and 4–5 September 2023). Data were reported as means ± standard deviation. Factorial ANOVA with three independent variables was performed (data is normally distributed and has homogeneous variance). Comparisons between means between groups were performed with Duncan`s multiple range test. Differences were considered significant when p < 0.05. Microsoft Excel 2021 was used for graphical representation.

3. Results and Discussions

3.1. Attack Severity Analysis of Diplocarpon rosae Wolf on the Rose Leaves

Damage due to Diplocarpon rosae Wolf in rose leaves from the rosary in the ‘Alexandru Buia’ Botanical Garden in Craiova ranged from minimal lesions (spots) on foliage to loss of some leaves (weakening the plant). The disease symptoms first appeared on the older leaves on the lower area of plants, and later the symptoms spread upwards. On the superior side of leaves, black spots were formed. They were small at the beginning, with radial aspect, and with no precise limited margins (Figure 1). In some cases, one noticed a brown coloration of the tissues on the inferior side of the leaves. The infected area of the leaves turned yellow around the spots, resulting in premature leaf fall. Severe infection can result in complete defoliation and dieback of the infected plants, but this did not happen in the botanical garden in Craiova as a result of the treatment applied to roses immediately after the appearance of the disease. However, roses with severe levels of infection produced less new growth and fewer blooms. The status of the health of the foliage ultimately impacts the health and longevity of the plant.

Data presented in

Table 1. The behavior of some rose varieties exposed to attack by Diplocarpon rosae Wolf.

Cultivar	Phytosanitary Status	Length (cm)	Width (cm)	Attack Degree (%)	Resistance Class
‘Orient Express’	Unattacked	7.2 ± 1.1	4.8 ± 0.7	30	Very sensitive (FS)
	Attacked	5.7 ± 1.2	4.2 ± 1.1
	Diminished appropriation (%)	20.83	12.5
‘Paul McCartney’	Unattacked	6.2 ± 1.2	4.1 ± 1.3	15	Very sensitive (FS)
	Attacked	6.0 ± 1.6	3.7 ± 1.8
	Diminished appropriation (%)	3.22	9.76
‘Terracotta’	Unattacked	7.6 ± 1.6	4.9 ± 1.2	20	Very sensitive (FS)
	Attacked	5.2 ± 1.4	4.2 ± 1.4
	Diminished appropriation (%)	31.58	14.28
‘Glorious’	Unattacked	5.9 ± 1.5	4.1 ± 0.4	35	Very sensitive (FS)
	Attacked	5.1 ± 0.9	3.9 ± 1.2
	Diminished appropriation (%)	13.55	4.88
‘Mythos’	Unattacked	8.20 ±1.8	5.1 ± 1.2	35	Very sensitive (FS)
	Attacked	7.15 ± 1.1	4.3 ± 1.0
	Diminished appropriation (%)	12.80	15.69
‘Holstein Perle’	Unattacked	6.6 ± 1.9	4.2 ± 1.3	20	Very sensitive (FS)
	Attacked	6.0 ± 1.2	3.6 ± 1.1
	Diminished appropriation (%)	9.1	14.28
‘Melina’	Unattacked	7.6 ± 1.3	6.4 ± 1.6	40	Very sensitive (FS)
	Attacked	5.7 ± 1.2	4.9 ± 1.5
	Diminished appropriation (%)	25.00	23.43
‘Asja’	Unattacked	6.2 ± 1.6	4.0 ± 1.3	9.2	Sensitive (S)
	Attacked	3.7 ± 1.8	2.9 ± 1.8
	Diminished appropriation (%)	40.32	27.5
‘Velvet Fragrance’	Unattacked	5.2 ± 1.6	3.2 ± 1.3	30	Very sensitive (FS)
	Attacked	4.1 ± 1.6	2.6 ± 0.9
	Diminished appropriation (%)	21.15	18.75
‘Cluj 2010′	Unattacked	4.5 ± 0.5	3.1 ± 0.5	40	Very sensitive (FS)
	Attacked	4.2 ± 0.9	2.5 ± 0.7
	Diminished appropriation (%)	6.67	19.34

illustrate that the attack of the fungus Diplocarpon rosae Wolf on roses grown in the rosary in the ‘Alexandru Buia’ Botanical Garden, Craiova in the summer of 2023 was strong, the calculated values of the attack degree oscillating between 9.2% (‘Asja’) and 40% (‘Melina’ and ‘Cluj 2010′). The average value of the attack degree in the ten cultivars of roses is 26.92%, the result indicating the constant presence of this pathogenic fungus all over the research area. It can be noted that in the climatic conditions experienced this year, one of the studied cultivars (‘Asja’) was susceptible to the phytoparasite attack while the other nine cultivars were very sensitive.

Leaf black spot on roses produced by the fungus Diplocarpon rosae Wolf affected the plants in all cultivars analyzed where the assessment was performed, but the differences between those cultivars highlighted the sensitivity of the relationship between plants and pathogens when exposed to some microclimatic environment factors. Diplocarpon rosae Wolf is a very dangerous pathogen because it causes premature aging of the roses. Also, this pathogen severely damages the young shoots by exposing them to frost because the wood of these shoots presents deep cracks where water will freeze, the effect accentuated by the premature defoliation of the affected leaves.

Our findings are similar to the results revealed by Yasin et al. (2016) [26], who found that the maximum means for rose black spot disease incidence in Pakistan was 56%, while the minimum threshold was 43.6%. Their research was performed in 40 rose gardens in Lahore, Kasur, Chakwal, and Rawalpindi districts of Punjab province, Pakistan during the spring seasons of 2012 and 2013. Knight and Wheeler (1978) [27] also found variation in rose susceptibility to different Diplocarpon rosae isolates.

FTIR spectroscopy was used to study diseased leaves comparatively with healthy rose leaves. The results of the ATR-FTIR spectra for the healthy plants and diseased plants (Figure 2) revealed the existence of various chemical constituents in leaves (

Table 2. ATR-FTIR spectra vibrational assignments for control (healthy) and diseased rose leaves.

Frequencies (cm−1)
Healthy Leaves	Diseased Leaves	Peak Assignment
3290	3292	νO–H, N–H
2915	2915	νasym C–H
2848	2848	νsym C–H
1731	1726	νC=O stretching due to lipids
1626	1625	νC=O, Amide I, stretching of protein
1462	1462	δasym CH2
1369	1376	Deformation N–H, δ CH2
1315	1321	νsym COO− stretching mainly from pectin
1242	1235	ν C–O from carbohydrates and lignin
1146	1152	νasym O–C–O stretching of polysaccharides
1102	1100	Stretching PO2− symmetric (phosphate II)
1026	1029	νC–O stretching vibration coupled with C–O bending of the C–OH groups of carbohydrates
687	685	CH out-of-plane bending vibrations

).

To compare disease-affected leaves to the control leaves (healthy leaves), peak assignments for observed bands were made (Table 2 and

Table 3. ATR-FTIR spectra vibrational assignments for healthy and diseased (with bold) rose leaves.

Frequencies (cm−1)
‘Orient Express’	‘Paul McCartney’	‘Terracotta’	‘Glorious’	‘Mythos’	‘Melina’	‘Asja’	‘Velvet Fragrance’	‘Cluj 2010′	Peak Assignment
3353 3362	3359 3344	3329 3317	3316 3334	3312 3307	3329 3318	3327 3334	3326 3325	3290 3291	Stretching O–H, N–H, C–H, N–H asymmetric (asym) [28]
2915 2915	2915 2915	2916 2915	2915 2915	2916 2915	2915 2915	2915 2915	2915 2916	2915 2915	νasym C–H
2848 2848	2848 2848	2848 2848	2848 2848	2848 2848	2848 2848	2848 2848	2848 2849	2848 2848	νsym C–H
1732 1732	1732 1731	1731 1730	1731 1731	1726 1730	1731 1730	1733 1731	1732 1730	1731 1731	νC=O stretching due to lipids
1629 1626	1613 1633	1626 1615	1626 1622	1630 1634	1620 1625	1625 1627	1625 1600	1626 1614	νC=O, Amide I, stretching of protein
1462 1462	1462 1462	1462 1462	1462 1462	1462 1461	1462 1461	1462 1462	1462 1461	1464 1461	δasym CH2
1375	1375 1375	1370	1373	1370 1366	1373 1372	1375 1369	1374 1371	1369	Stretching C–N [28] Deformation N–H, δ CH2 [29]
1315 1314	1315 1317	1315 1316	1315 1314	1323 1310	1315 1310	1315 1316	1315 1313	1315 1318	Amide III band components of proteins [30]
1240 1234	1240 1236	1242 1240	1241 1233	1236 1233	1240 1234	1241 1234	1241 1233	1242 1238	νC–O from carbohydrates and lignin
1167 1145	1165 1148	1159 1159	1166 1146	1160 1159	1147 1144	1147 1144	1147 1149	1146 1164	νasymO–C–O stretching of polysaccharides
1101	1101	1100	1102	1100	1099	1101	1099	1100	Stretching PO2− symmetric (phosphate II)
1028 1029	1021 1026	1030 1036	1021 1031	1030 1031	1025 1032	1019 1026	1029 1029	1026 1031	νC–O stretching vibration coupled with C–O bending of the C–OH groups of carbohydrates
718 716	717 716	718 718	718 717	714 713	718 716	718 718	718 718	718 718	out-of-plane bending vibrations

). NH absorption was observed around 3350-–3290 cm⁻¹. Bands appearing in the region of 2920–2918 and 2851–2850 cm⁻¹ were assigned to the asymmetric (ν_asym) and symmetric (ν_sym) CH₂ stretching vibrations.

The infrared spectra of protein are characterized by a set of absorption regions known as the amide region and the C–H region. Amide I band appears at 1625–1629 cm⁻¹ as a very strong band. In diseased leaves, this band appears at low wavenumber. In addition, the peak at 1729–1731 cm⁻¹ assigned to the C=O stretching vibration means that there are some carbonyl compounds (wax esters and ketones) or carboxylic acid groups.

Some attention was also paid to the amide bands (1626 cm⁻¹ and 1315 cm⁻¹) and to the C–O stretching region between 1200 and 1000 cm⁻¹. The bands corresponding to the stretching vibrations of polysaccharides (ν_asymO–C–O) appeared at a lower wavenumber in the diseased leaves (Table 2 and Table 3). Amide III bands from around 1315 cm⁻¹ are mainly associated with CN stretching and NH bending vibrations and increase in intensity in affected leaves.

A band at 687 cm⁻¹ which appeared in all samples is attributed to the methylene rocking vibration νCH₂.

The comparison between the FTIR spectrum of healthy rose leaves and the diseased leaves reveals a clear difference in the carbohydrates that are present in each plant (Table 2 and Figure S1).

3.2. Influence of the Phytosanitary Status, Cultivar, and Harvest Time on the Phenolic, Chlorophyll, and Alkaloid Content of Rose Leaves

Table 4. Variations in the content of phenolic compounds (TPC), tannins (TTC), flavonoids (TFC), alkaloids, and chlorophyll in the rose leaves depending on the date of analysis. Data are presented as mean ± SD (standard deviation).

Biochemical Parameters	Time	Phytosanitary Status	Cultivar
			‘Orient Express’	‘Paul McCartney’	‘Terracotta’	‘Glorious’	‘Mythos’	‘Holstein Perle’	‘Melina’	‘Asja’	‘Velvet Fragrance’	‘Cluj 2010′
TPC	28–29 June	Healthy	980.17 ± 0.20	889.20 ± 0.85	984.85 ± 3.19	975.41 ± 1.28	1151.75 ± 0.97	1020.77 ± 3.49	1237.65 ± 2.64	1147.90 ± 0.43	1020.98 ± 0.02	1090.65 ± 0.67
		Affected	985.13 ± 1.28	929.35 ± 1.02	1017.98 ± 3.70	953.73 ± 1.89	1364.24 ± 4.59	1091.76 ± 2.73	1269.07 ± 5.45	1152.61 ± 2.30	1105.25 ± 7.57	1249.14 ± 5.11
	12–13 July	Healthy	959.32 ± 0.63	867.76 ± 0.59	950.73 ± 0.74	929.04 ± 1.38	998.13 ± 0.69	959.32 ± 0.63	1074.53 ± 1.66	1040.09 ± 0.70	932.78 ± 0.29	999.96 ± 1.53
		Affected	962.01 ± 0.54	887.83 ± 1.77	976.60 ± 0.84	946.53 ± 0.40	1141.84 ± 0.36	984.85 ± 3.19	1099.61 ± 2.58	1052.01 ± 3.09	965.71 ± 1.25	1076.78 ± 6.69
	4–5 September	Healthy	709.49 ± 1.31	784.79 ± 0.97	873.12 ± 1.10	829.25 ± 1.07	833.28 ± 0.39	821.04 ± 0.50	813.59 ± 0.73	908.82 ± 1.60	844.15 ± 0.32	889.59 ± 1.19
		Affected	744.83 ± 1.12	813.79 ± 0.62	886.49 ± 1.12	852.34 ± 0.91	936.28 ± 2.04	847.13 ± 0.61	823.44 ± 0.62	926.41 ± 1.15	864.89 ± 0.83	925.98 ± 1.74
TTC	28–29 June	Healthy	375.14 ± 0.11	389.83 ± 0.10	387.28 ± 0.08	368.57 ± 0.64	371.98 ± 0.54	371.87 ± 0.54	406.99 ± 0.06	374.14 ± 0.08	379.77 ± 0.48	372.09 ± 0.15
		Affected	427.60 ± 0.08	397.19 ± 0.07	407.88 ± 0.06	384.42 ± 0.06	393.02 ± 0.16	386.50 ± 0.02	418.02 ± 0.12	396.49 ± 0.04	415.86 ± 0.14	393.79 ± 0.52
	12–13 July	Healthy	353.24 ± 0.06	380.49 ± 0.15	369.42 ± 0.57	343.32 ± 0.08	348.61 ± 0.94	356.58 ± 0.53	421.92 ± 57.71	357.92 ± 0.85	363.87 ± 0.38	357.25 ± 0.32
		Affected	371.35 ± 0.14	386.71 ± 1.09	398.78 ± 1.14	357.11 ± 0.22	369.82 ± 0.08	374.91 ± 0.58	395.25 ± 0.64	387.29 ± 0.41	395.42 ± 0.22	376.55 ± 1.23
	4–5 September	Healthy	313.50 ± 0.19	353.14 ± 0.06	335.72 ± 0.59	323.93 ± 0.16	327.38 ± 0.01	332.46 ± 0.60	355.53 ± 0.70	340.66 ± 0.60	352.33 ± 0.48	340.97 ± 0.33
		Affected	336.65 ± 0.22	366.99 ± 0.04	357.25 ± 0.11	326.73 ± 0.07	331.48 ± 0.07	349.94 ± 0.61	362.65 ± 0.56	356.34 ± 1.16	365.24 ± 0.58	345.79 ± 0.50
TFC	28–29 June	Healthy	285.05 ± 1.78	305.04 ± 4.37	193.87 ± 3.55	280.47 ± 3.94	337.41 ± 4.07	327.63 ± 2.91	321.06 ± 1.65	316.09 ± 3.66	358.25 ± 1.45	346.83 ± 1.41
		Affected	375.94 ± 4.19	366.79 ± 4.21	224.04 ± 3.09	364.49 ± 2.81	377.83 ± 1.92	372.19 ± 4.01	347.45 ± 3.90	371.46 ± 2.99	376.35 ± 4.97	391.29 ± 3.82
	12–13 July	Healthy	270.76 ± 3.46	274.95 ± 2.15	186.53 ± 2.89	248.77 ± 2.53	310.03 ± 2.01	288.95 ± 1.42	282.04 ± 1.49	279.84 ± 3.24	321.83 ± 1.63	332.31 ± 3.47
		Affected	345.99 ± 2.17	293.98 ± 1.49	201.46 ± 1.79	361.72 ± 3.25	341.12 ± 1.96	312.57 ± 1.45	330.49 ± 1.56	346.25 ± 4.25	350.96 ± 2.80	367.48 ± 2.17
	4–5 September	Healthy	204.13 ± 0.42	199.13 ± 1.60	152.55 ± 1.87	226.94 ± 1.62	224.65 ± 2.69	225.55 ± 1.67	227.34 ± 1.95	263.69 ± 2.35	292.33 ± 1.32	309.63 ± 1.82
		Affected	253.29 ± 1.28	239.77 ± 2.90	156.60 ± 1.82	238.79 ± 1.42	229.79 ± 1.64	261.57 ± 2.29	252.38 ± 2.43	260.19 ± 2.04	331.06 ± 4.24	317.72 ± 4.93
Chlorophyll a + b	28–29 June	Healthy	270.69 ± 1.60	288.73 ± 1.24	239.62 ± 1.11	322.73 ± 1.17	321.40 ± 1.04	459.72 ± 5.80	293.27 ± 0.57	250.27 ± 0.60	212.73 ± 0.75	283.08 ± 0.89
		Affected	65.06 ± 0.91	133.72 ± 0.19	95.64 ± 0.10	101.00 ± 0.47	222.61 ± 4.56	310.86 ± 5.34	212.51 ± 2.44	176.92 ± 0.09	88.78 ± 2.62	207.31 ± 0.48
	12–13 July	Healthy	144.47 ± 5.45	228.17 ± 0.70	150.24 ± 0.82	232.07 ± 0.41	192.49 ± 0.75	321.45 ± 0.99	237.49 ± 5.39	211.99 ± 5.24	151.02 ± 0.22	187.09 ± 5.28
		Affected	34.05 ± 0.02	118.63 ± 0.22	66.46 ± 1.00	58.75 ± 0.27	164.26 ± 5.67	210.20 ± 0.63	137.84 ± 0.43	161.71 ± 0.83	79.66 ± 1.70	148.30 ± 0.08
	4–5 September	Healthy	93.09 ± 0.67	136.84 ± 4.21	117.02 ± 4.71	104.80 ± 5.87	96.93 ± 0.57	99.60 ± 0.43	134.82 ± 0.32	81.30 ± 3.95	58.69 ± 0.20	123.26 ± 3.00
		Affected	28.44 ± 0.35	63.07 ± 2.58	25.31 ± 0.01	23.40 ± 0.11	57.37 ± 0.33	46.82 ± 0.16	61.35 ± 0.26	75.61 ± 0.70	32.21 ± 0.24	111.47 ± 4.51
Alkaloids	28–29 June	Healthy	78.50 ± 0.05	86.41 ± 0.09	87.90 ± 0.11	79.22 ± 0.05	88.24 ± 0.05	82.90 ± 0.56	88.07 ± 0.02	75.88 ± 0.06	80.12 ± 0.31	82.02 ± 0.01
		Affected	89.16 ± 0.09	91.45 ± 0.02	89.58 ± 0.04	80.08 ± 0.03	94.62 ± 0.09	85.97 ± 0.13	90.57 ± 0.07	78.85 ± 0.05	85.53 ± 0.06	89.42 ± 0.08
	12–13 July	Healthy	88.99 ± 0.05	91.01 ± 0.10	89.17 ± 0.16	88.02 ± 0.09	92.42 ± 0.08	89.16 ± 0.07	92.83 ± 0.04	87.83 ± 0.09	85.81 ± 0.02	86.66 ± 0.01
		Affected	92.97 ± 0.10	95.92 ± 0.11	91.17 ± 0.16	92.16 ± 0.04	92.88 ± 0.10	91.69 ± 0.07	93.55 ± 0.09	90.71 ± 0.09	91.58 ± 0.03	92.55 ± 0.03
	4–5 September	Healthy	93.31 ± 0.13	93.86 ± 0.15	97.13 ± 0.18	94.07 ± 0.03	96.21 ± 0.06	90.10 ± 0.09	94.09 ± 0.09	89.42 ± 0.05	90.79 ± 0.05	89.76 ± 0.11
		Affected	98.25 ± 0.13	96.53 ± 0.16	97.70 ± 0.13	97.82 ± 0.08	99.05 ± 05	92.17 ± 0.11	94.91 ± 0.09	97.66 ± 0.04	94.52 ± 0.15	95.28 ± 0.06

shows the levels of phenolic compounds (TPC), tannins (TTC), flavonoids (TFC), chlorophyll, and alkaloids, determined in the leaves of ten rose cultivars depending on their phytosanitary status, at three moments of analysis.

As an overview, a comparison between the healthy leaves of all cultivars at the first moment of analysis (28–29 June 2023) indicated that the group consisting of ‘Melina’ (1237.65 mg GAE 100 g⁻¹), ‘Mythos’ (1151.75 mg GAE 100 g⁻¹), ‘Asja’ (1147.90 mg GAE 100 g⁻¹), ‘Cluj 2010’ (1090.65 mg GAE 100 g⁻¹), ‘Velvet Fragrance’ (1020.98 mg GAE 100 g⁻¹), and ‘Holstein Perle’ (1020.77 mg GAE 100 g⁻¹) presented the highest values of TPC, but also presented the highest values of TFC (358.25 mg CE 100 g⁻¹, ‘Velvet Fragrance’; 346.83 mg CE 100 g⁻¹, ‘Cluj 2010’; 337.41 mg CE 100 g⁻¹, ‘Mythos’; 327.63 mg CE 100 g⁻¹, ‘Holstein Perle’; 321.06 mg CE 100 g⁻¹, ‘Melina’; and 316.09 mg EC 100 g⁻¹ ‘Asja’). In addition, similar to ‘Terracotta’ (387.28 mg GAE 100 g⁻¹) and ‘Paul McCartney’ (389.83 mg GAE 100 g⁻¹), ‘Melina’ cv. stood out as having the highest level of tannins, 406.99 mg GAE 100 g⁻¹. Last but not least, the tannins fraction dominated the phenolic compounds profile compared to flavonoids (the tannins/flavonoids ratio ranged from 1.06, in ‘Velvet Fragrance’ cv., to 2.00, in ‘Terracotta’ cv.).

The results obtained in this study regarding TPC are comparable to those reported by Nowak and Gawlik-Dziki (2007) [31] for the leaves from 17 Rosa L. cultivars in the Lublin region of Poland, harvest in June 2003 during the florescence of plants. Low levels of TPC were found in R. rugosa (570 mg GAE 100 g⁻¹) and R. vosagiaca (770 mg GAE 100 g⁻¹), whereas R. canina var. dumalis and other R. canina species (except for R. canina var. canina, with 990 mg GAE 100 g⁻¹) contained the highest amounts of phenolics in leaves (1220 to 1520 mg GAE 100 g⁻¹). Cunja et al. (2014) [32] have reported more than 50 phenolic compounds (belonging to the subclasses of flavonols, phenolic acids, and their derivatives) present in leaves of four rose species (R. glauca, R. canina, R. sempervirens, and R. rubiginosa) and three cultivars (Rosarium Uetersen, Ulrich Brunner Fils, and Schwanensee) grown in the botanical garden in Ljubljana. The TFC values of rose leaves in our study were lower than those reported for different Rosa sp. species by D’Angiolillo et al. (2018) [33] and Ozsoy et al. (2013) [34]. As in our study, the authors indicated a moderate influence based on cultivar and little differences between TFC values determined at different harvesting periods of leaves.

The highest total chlorophyll content in the healthy leaves was found for ‘Holstein Perle’ cv. (459.72 mg 100 g⁻¹), while the lowest values were found in the healthy leaves of ‘Velvet Fragrance’ (212.73 mg 100 g⁻¹) and ‘Terracotta’ (239.62 mg GAE 100 g⁻¹). In our study, the total chlorophyll content was higher than those levels reported by D’Angiolillo et al. (2018) [33] in the leaves of four Sicilian roses harvested during two different periods (June and October). As in the present study, their results showed that the total chlorophyll content was higher in the leaves of all species harvested in June (92 mg 100 g⁻¹, on average) than in those harvested in October (76 mg 100 g⁻¹, on average). Differences among the species were higher in June (from 76 to 111 mg 100 g⁻¹ for R. micrantha and R. corymbifera, respectively) and lower in October (from 66 to 83 mg 100 g⁻¹ for R. micrantha and R. sempervirens, respectively). In addition, Sparinska and Rostoks (2012) [35] reported high variations (from 134 to 193 mg 100 g⁻¹) in the leaf chlorophyll content of R. rugosa cultivars in Latvia and they mainly associated these differences with the genotype.

The cultivars with alkaloid content in the healthy leaves exceeding 88 mg AE 100 g⁻¹ were ‘Melina’ and ‘Mythos’ (with 88.07 and 88.24 mg AE 100 g⁻¹, respectively), while the smallest values were found in the cultivars ‘Asja’ (75.88 mg AE 100 g⁻¹) and ‘Orient Express’ (78.50 mg AE 100 g⁻¹).

Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7 graphically present the variation in the contents of TPC, TTC, TFC, total chlorophyll, and alkaloids recorded for the healthy and affected rose leaves of the studied cultivars at the three moments of analysis.

Without exception, a tendency for the TPC and total chlorophyll content to decrease during the harvest period could be observed for both healthy and unhealthy plants (Figure 3 and Figure 6).

In the case of TPC, in the group of healthy plants the strongest oscillation between the first and last moment of analysis was observed for ‘Melina’ and ‘Mythos’ cvs., followed by ‘Orient Express’, ‘Asja’, and ‘Cluj 2010’. A similar reduction in TPC content was also observed in the group of unhealthy plants, mainly for ‘Melina’ and ‘Mythos’ cvs., followed by ‘Cluj 2010’ and ‘Orient Express’. At the opposite pole, the smallest differences in TPC between the first and the last moment of harvest were observed for ‘Paul McCartney’ and ‘Terracotta’ cvs. (in both groups of plants, healthy and unhealthy).

With only one exception (‘Melina’ cv., healthy plants, second harvest time), TTC decreased continuously between the three harvest times, and the highest TTC reduction in the group of healthy plants was recorded for ‘Orient Express’, ‘Terracotta’, and ‘Melina’ cvs. An even more pronounced TTC reduction was observed in unhealthy plants of the ‘Orient Express’ cultivar, as well as in those of ‘Mythos’, ‘Glorious’, and ‘Melina’ cvs. The lowest tannin losses were shown by the healthy plants of ‘Velvet Fragrance’ and ‘Cluj 2010’ cvs., followed by ‘Asja’, and in the group of sick plants, by the ‘Paul McCartney’ cv.

‘Mythos’ cv., followed by ‘Paul McCartney’, had the greatest loss in terms of the leaves’ flavonoid content (in both groups of plants, healthy and diseased). Also, significant reductions in TFC were recorded in unhealthy specimens of ‘Glorious’ and ‘Orient Express’ cvs. Small differences between the first and the last harvest time were observed for ‘Cluj 2010’, ‘Terracotta’ (healthy plants), and ‘Velvet Fragrance’ (unhealthy plants) cvs.

The most intense losses of total chlorophyll content were observed for ‘Holstein Perle’ cv., followed by ‘Mythos’ (healthy and diseased plants), while those with the lowest levels of total chlorophyll were the unhealthy plants of the ‘Orient Express’ cvs., followed by ‘Velvet Fragrance’.

Alkaloids were the only chemicals in this study whose level increased between the first and the last harvest time, the greatest increase being found for ‘Glorious’ and ‘Asja’ cvs., but with increases also observed for ‘Orient Express’ and ‘Velvet Fragrance’ (healthy and unhealthy plants). The lowest oscillation of alkaloids was observed for ‘Melina’ and ‘Mythos’ cv. unhealthy plants. This behavior is explainable because, in plants, alkaloids are secondary metabolites produced in response to environmental modulations and biotic or abiotic stress, which gives them structural diversity and significant biological activities [36,37]. The growth of leaf alkaloid content with the increasing age of the plant was observed in all species [38,39,40,41]. Since the availability of nitrogen (N) is expected to play an important role in the biosynthesis and accumulation of alkaloids in plants, the content of alkaloids is further increased in diseased plants.

As presented in

Table 5. The significance of the influence of the cultivar, harvest time, and phytosanitary status on the phenolic, tannin, flavonoid, chlorophyll, and alkaloid content of rose leaves samples.

Investigated Factor	TPC (mg GAE 100 g−1)	TFC (mg CE 100 g−1)	TFC (mg CE 100 g−1)	Chlorophyll a + b (mg 100 g−1)	Alkaloids (mg AE 100 g−1)
Cultivar	0.000	0.000	0.000	0.000	0.000
Harvest time	0.000	0.000	0.000	0.000	0.000
Phytosanitary status	0.000	0.000	0.000	0.000	0.000
Cultivar x Harvest time	0.000	0.000	0.000	0.000	0.000
Cultivar x Phytosanitary status	0.000	0.000	0.000	0.000	0.000
Harvest time x Phytosanitary status	0.000	0.002	0.000	0.000	0.000
Cultivar x Time x Phytosanitary status	0.000	0.000	0.000	0.000	0.000

p values are presented according to the ANOVA analysis of variance (significant at 0.05 level).

, the phenolic, tannin, flavonoid, chlorophyll, and alkaloid content was significantly influenced by the cultivar, the harvest time, the phytosanitary status, and their interaction.

The correlations between the total content of the five classes of compounds and the degree of attack (AD) from the leaves of the ten cultivars of Rosa sp. are summarized in

Table 6. Correlation matrix between the degree of attack (AD), the content of phenolic compounds (TPC), tannins (TTC), flavonoids (TFC), chlorophyll a + b, and alkaloids in the rose leaves (r values are presented).

		AD (%)	TPC (mg GAE 100 g−1)	TTC (mg GAE 100 g−1)	TFC (mg CE 100 g−1)	Chlorophyll a + b (mg 100 g−1)	Alkaloids (mg AE 100 g−1)
AD	Pearson correlation	1	−0.107	−0.189 *	0.073	−0.517 ***	0.510 ***
	Sig. (2-tailed)		0.154	0.011	0.327	0.000	0.000
TPC	Pearson correlation		1	0.636 ***	0.629 ***	0.506 ***	−0.403 ***
	Sig. (2-tailed)			0.000	0.000	0.000	0.000
TTC	Pearson correlation			1	0.516 ***	0.279 ***	−0.339 ***
	Sig. (2-tailed)				0.000	0.000	0.000
TFC	Pearson correlation				1	0.280 ***	−0.409 ***
	Sig. (2-tailed)					0.000	0.000
Chlorophyll a + b	Pearson correlation					1	−0.604 *** 0.000
	Sig. (2-tailed)

* Correlation is significant at the 0.05 level; and *** the correlation is significant at the 0.001 level.

. As shown in this table, the correlations between attack degree (AD) and tannins, total chlorophyll, or alkaloids were significant; they were negative for tannins (r = −0.189 *) and total chlorophyll (r = −0.517 ***) and positive for alkaloids (r = 0.510 ***). All correlations established between TPC, TTC, TFC, and total chlorophyll were positive and strongly significant. In the case of alkaloids, negative and very significant correlations were established with TPC (r = −0.403 ***), TTC (r = −0.339 ***), TFC (r = −0.409 ***), and total chlorophyll (r = −0.604 ***), respectively.

4. Conclusions

Our study evaluated the influence of phytosanitary status, cultivar, and harvest time on the phenolic, chlorophyll, and alkaloid content of Rosa sp. leaves.

In June 2023, in the rosary of the ‘Alexandru Buia’ Botanical Garden in Craiova, which is managed as an organic system, the presence of the Diplocarpon rosae Wolf was detected. The disease, called ‘black spot of rose’, is one of the diseases known for the damage it produces in this crop; it occurs naturally and affected all ten rose cultivations from the nursery sector. The attack of this pathogenic fungus on the ten rose cultivars was strong, with the calculated values of the attack degree oscillating between 9.2% (‘Asja’ cv.) and 40% (‘Melina’ and ‘Cluj 2010′ cvs.), with an average value of 26.92%. Only one of the studied cultivars (‘Asja’) was susceptible to the Diplocarpon rosae Wolf attack, while the other nine cultivars were very sensitive.

The treatment scheme (a mixture of Faster 10 CE, Ditan, and Nissorun 10 WP) conducted on 7 July 2023 stopped the disease spread in the entire area. Considering the severity of the attack produced by the fungus Diplocarpon rosae Wolf on the rose leaves in 2023, the management of the ‘Alexandru Buia’ Botanical Garden in Craiova decided that the best way to fight this disease would be preventive treatment. Due to the weather conditions in spring, with rains and high humidity, it must be taken into account that all rain that falls after the application of the fungicides will significantly reduce the protection they offer. In addition, the rain is brought by air currents, which, in addition to moisture, will also transport seeds. Despite the effectiveness of the treatment scheme applied this year to limit the spread of the disease, we appreciate that extensive research is needed on this topic, and that this is important in terms of the specifics of the latest climate change conditions and the particularities of rose plants. Fungal diseases are best managed through a multidisciplinary approach that includes plant selection, planting time, level of fertility, sanitation, and applications of fungicides. Some roses exhibit a greater tolerance than others as regards the attack of fungal diseases. Their different susceptibility to disease will dictate the management practices that must be employed to maintain the health of the rose garden.

The comparison of FTIR spectra of the healthy with those of the diseased leaves pointed out a clear difference in the carbohydrates that are present in each plant. The carbohydrate region (between 1000 and 1240 cm⁻¹) is the region that was shown to be the most significant in terms of identifying virus diseases.

The results of this work show that rose leaves accumulate large amounts of phenolic compounds, these compounds having antioxidant and free radical scavenging abilities, with potential effects on human health. This finding highlights the need to include the edible rose in the human diet.

Special attention was paid to the influence of the cultivar and harvest time on the phenolic, chlorophyll, and alkaloid content of Rosa sp. leaves correlated to the phytosanitary status of roses. Our study indicated that between attack degree and tannins, total chlorophyll or alkaloids settled the significant correlations, negative for tannins (r = −0.189 *) and total chlorophyll (r = −0.517 ***) and positive for alkaloids (r = 0.510 ***). In addition, all correlations between phenolic compounds, tannins, flavonoids, and total chlorophyll were positive and very significant. In the case of alkaloids, negative and very significant correlations were established with TPC (r = −0.403 ***), TTC (r = −0.339 ***), TFC (r = −0.409 ***), and total chlorophyll (r = −0.604 ***), respectively.