AAAS_41_2_100-108

Introduction

Rose (Rosa indica), a woody evergreen flower shrub, that belongs to the family Rosaceae and genus Rosa (Rasheed et al., 2015). It is one of the largest flower banks in an ecosystem having about 200 native species and greater than 18000 cultivars that have originated from North America, Asia, Northwest Africa, and Europe (Ali et al., 2018; Sazzad et al., 2022; Desta et al., 2022). R. centifolia L., R. gruss-an-teplitz, R. damascena, R. bourboniana and R. gallica are the most significant species of the genus Rosa; among all, R. gruss-an-teplitz (class: Bourbon), locally termed as red rose, is the second most widely cultivated species after R. damascena (Nawaz et al., 2011). It is a very popular flower in Pakistan and has significant value in our society, mostly used as a sign of love and charm, both in peace and in war (Leghari et al., 2016; Mansha et al., 2021). Globally, roses are cultivated on an area of 60,447 ha, where India, China, Ecuador, Netherland, Kenya and Ethiopia are the major producers of the crop. In Pakistan it is cultivated on an approximate area of 607.04 ha, as cut flowers, in a diverse climate from dry to humid and hot to cold regions (Qaisar et al., 2023; Yasin et al., 2016; Shah et al., 2021).

Black spot disease impedes the cultivated rose production, and is one of the most drastic and devastating diseases of field grown roses caused by an ascomycete fungus Diplocarpon rosae (whose imperfect stage is Marssonina rosae) (Yeluguri et al., 2022; Debener, 2019). This pathogen is specifically associated with roses; however other species of Diplocarpon may also affect various members of the family Rosaceae such as D. mali (infecting apple Malus domestica), D. mespilli (infecting pear Pyrus communis L. and quince Cydonia oblonga), D. earlianum (infecting strawberries Fragaria × ananassa) and M. brunnea (infecting poplar trees) (Debener, 2019).

The characteristic symptoms of this disease includes black spots having asymmetrical borders formed on the adaxial surface of leaflet, accompanied by the yellowing of leaves, premature leaf fall, loss of strength and also loses its aesthetic quality (Marolleau et al., 2020). Initially the fungus spreads mainly through two celled asexual spores (conidia) by rain water and these spores can over winter on dead leaves and stem tissues. Then the commencement of disease occurs in the early spring season when the conditions are favorable for spore germination and infection (Münnekhoff et al., 2017). Conidia of D. rosae are colorless that germinate quickly in water or on the artificial agar media, but the mycelial growth of fungus is tremendously slow on media (Yeluguri et al., 2022). Therefore, it is problematic, especially in the areas of high annual rainfall and humidity (Lopez Arias et al., 2020).

The infection process begins when the spores germinate and develop a germ tube with an appressorium. Mainly from the bi-cellular conidia, germ tube emerges from the larger cells, but in most cases two germ tubes one from each conidia can also be observed. The pathogen then penetrates the leaf cuticle and begins to form subcuticular and intercuticular hyphae. In addition to this, the inter-cellular hyphae infect both epidermal and parenchymal cells within first 24 hours after penetration and thus form a prominent haustoria (Debener, 2019). Haustoria are observed during the first stage of infection mainly for obtaining nutrients. Then the transition of pathogen occurs from biotrophic lifestyle to necrotrophic one, forming necrotrophic intercellular hyphae and producing acervuli and second-generation conidia (Lopez Arias et al., 2020). Symptoms may appear on susceptible plants within four days after spore germination. Ideal temperature for spore germination ranges between 0 to 33 °C (Mihaescu and Din, 2019; Debener, 2019).

Management of plant diseases primarily depends on an intensive use of synthetic chemicals (Marolleau et al., 2020). As Poole and Arnaudin (2014) disclosed that fungicides are the critical component of integrated disease management strategies, serving as the last line of defense against plant diseases. While these do not directly increase crop yield, but play a crucial role in protecting the inherent yield potential that may be realized by growers in the absence of disease. These are used as bio-pesticides in both developed and developing countries. It is a quick, simple and direct method that directly affect the pathogens and provide quick response as compared to others that are considerably slow (Ahmad et al., 2020). The contemporary study was intended to diagnose the anatomical variations in plants due to infection of pathogen and it’s management using different fungicides

Materials and Methods

Isolation, purification and identification of pathogen

Infected rose leaves having typical black spot disease symptoms were collected from horticultural research area, University of Agriculture, Faisalabad and brought to laboratory for the isolation of causative agent. Infected leaves were washed thoroughly and cut down into small pieces, about 2-3 mm in diameter, then surface disinfected in laminar flow cabinet (RTVL-1312) by dipping in 1%, Sodium, Hypochlorite (NaOCl) solution for 15 seconds and then washed three times with sterilized distilled water and left for drying. Then at least three pieces were placed on petri plates containing autoclaved Potato Dextrose Agar (PDA) medium by using sterilized forceps, and incubated at 25oC for obtaining fungal growth. The targeted pathogen was purified by using single hyphal tip technique and then identified on the basis of morphological characters i.e., colony color, conidia shape, size, and septation (Matte et al., 2022).

Pathogenicity test

Pathogenicity test was performed under in-vivo conditions by inoculating the rose plants with Diplocarpon rosae spore suspension to confirm the association of pathogen with host plant. Fungal spore suspension was prepared in distilled water and adjusted as 1×105 conidia/ml-1 by using haemocytometer. Spore suspension was applied onto the rose plants by using spray method. Plants were sprayed with water day by day for maintaining high humidity. The symptoms were observed after one week of inoculation. Diseased samples were collected and brought to the laboratory. Following the Koch’s postulates, re-isolation was carried out from diseased plants. The re-isolated pathogen was same as observed in parent culture (Matte et al., 2022).

In-vitro evaluation of different fungicides against Diplocarpon rosae causing black spot disease of rose

Five different fungicides (Contaf, Canbinax, Agrosporter, Defeater plus and Fossil) were evaluated against Diplocarpon rosae following poisoned food technique. Potato dextrose agar (PDA) media was amended with three different (200, 300 and 400ppm) concentrations of fungicides. A 5mm disc from seven days old Diplocarpon rosae culture was cut with the help of sterilized Cork borer and placed with the help of spatula in the center of each petri plate containing PDA media incorporated with fungicides, while the control plates contained only PDA media. Then the petri plates were wrapped and incubated at 25˚C in an incubator (GEN2 BOD). The fungal growth was recorded in millimeter after 48 hours.

Assessment of different fungicides against black spot disease of rose caused by Diplocarpon rosae under field conditions

For in-vivo management, rose plants were grown in Horticulture research area at UAF. All the horticultural practices were implemented to keep healthy plants. 40 days old rose plants were firstly inoculated by spraying spore suspension on fresh leaves, then the most effective fungicides (Contaf and Canbinax) under lab condition were applied as combination and solo treatments using hand sprayer. The plants treated with distilled water were kept for control. Data regarding disease incidence percentage was recorded for three weeks with one week of interval using formula given by (Mansha et al., 2021).

Anatomical analysis

For anatomical analysis healthy, inoculated and treated leaf samples were collected from Horticultural field area accordingly. For long time preservation, samples were preserved in aceto-alcohol solution. Transverse sections (T.S) were cut by using double edge razor blade by free hand sectioning method. Thin sections were selected and dehydrated in consecutive ethanol grades (30, 50, 70, 90 and 100% ethanol) and then stained by using two biological stains (safranin and fast green). After staining sections were fixed on glass slide by using a drop of DPX and then visualized under camera-equipped digital compound microscope (MT4300-LV-HD, Meiji Techno, Japan). Following leaf parameters were studied i.e. midrib thickness (MT) (µm), adaxial epidermal thickness (ADE) (µm), abaxial epidermal thickness (ABE) (µm), vascular bundle area (VBA) (µm²) and parenchyma cell area (PCA) (µm²). Readings were recorded by using an ocular micrometer, which was calibrated by stage micrometer.

Statistical analysis

The data was analyzed by using statistical software “Statistics 8.1” at (ρ≤0.05). The data was subjected to analysis of variance (ANOVA), by using least significance difference (LSD) at alpha (0.05%).

Results

Isolation and morphological identification of pathogen

The colony of the isolated fungus was black in color and dichotomously branched that grew in a radial pattern around their emerging points. Whereas, microscopy showed colorless, bi-nucleate, aseptate and two celled conidia. Hyphae contained conidiogeneous cells at their tips, having two celled conidia that were separated from conidiophore through septum. By comparison with available literature, the isolated fungus was Diplocarpon rosae (Figure 1).

Pathogenicity test

Due to artificial inoculation using spore suspension, rose plants showed small, circular, purple colored spots at initial stage, while laterally these spots became larger, irregularly shaped and blackish in color, followed by yellowing of leaves. All these symptoms were same as observed earlier at the time of sampling (Figure 2).

Evaluation of chemical fungicides against Diplocarpon rosae causing black spot disease of rose under lab conditions

All the treatments showed significant disease control. Among all fungicides, Contaf showed minimum mycelial growth (4.87 mm) followed by Canbinax (5.41 mm), Fossil (6.35 mm), Defeater plus (7.88 mm) and Agrosporter (12.34 mm) as compared to the control plate (23.96 mm) (Figure 3a). The impact of interaction between fungicides and their concentrations indicated that Contaf showed minimum mycelial growth at all three concentrations such as 400 ppm (2.42 mm), 300 ppm (5.16 mm) and 200 ppm (7.04 mm), followed by Canbinax at 400 ppm (2.44 mm), 300 ppm (5.98 mm), and 200 ppm (7.811 mm). Whereas remaining treatments, Fossil (8.73, 4.61, 3.92 mm), Defeater plus (10.72, 7.58, 5.33 mm) and Agrosporter (14.83, 13.24, 8.94 mm) showed moderate mycelial growth at 200, 300 and 400 ppm concentration respectively (Figure 3b). Additionally, the interaction of fungicides with effect of time durations revealed that, Contaf was highly significant in inhibiting fungal growth, which expressed minimum fungal growth at day 1 (3.75 mm) and day 3 (5.87 mm) as compared to the remaining treatments, such as Canbinax, Fossil, Defeater plus and Agrosporter at day 1 (4.55, 5.65, 6.73 and 11.62 mm) and day 3 (6.25, 7.13, 9.46 and 13.45 mm) (Figure 3c).

Assessment of different fungicides for the management of black spot disease caused by Diplocarpon rosae under field conditions

Among all treatments, the mixture of Contaf + Canbinax showed minimum disease incidence (30.5%), followed by individual application of Contaf (36.6%) and Canbinax (41.6%) (Figure 4A), whereas, the data regarding interaction between fungicidal treatments and days interval indicated that the combination (Contaf + Canbinax) exhibited durable effect against black spot of rose with minimum disease incidence (24, 28.3, 43.3%) after 1st, 2nd and 3rd week of application, followed by solo application of Contaf (25, 35, 50%) and Canbinax (30, 38, 56%) respectively (Figure 4B).

Leaf anatomy

Anatomical analysis of leaves of Rosa gruss an teplitz from healthy and Diplocarpon rosae inoculated plants showed a diverse behavior of investigated parameters. Observations of targeted parameters among healthy plant leaves were midrib thickness (1744.32µm), adaxial epidermal thickness (62.98µm), abaxial epidermal thickness (44.21µm), vascular bundle area (2095796µm2) and parenchyma cell area (36974.48µm2), whereas in diseased leaves observations expressed enlarged midrib thickness (2156.17µm), shattered adaxial epidermal thickness (81.76µm), burst abaxial epidermal thickness (61.77µm), shrunk vascular bundle area (1566059µm2) and increased parenchyma cell area (124745.7µm2) (Figures 5 and 6). Moreover, the effect of pathogen on anatomical parameters in different inoculation stages indicated a gradual increase in measurement of all parameters including midrib thickness, adaxial epidermal thickness, abaxial epidermal thickness and parenchyma cell area except vascular bundle area (Figure 7), where inoculation stage three was found highly effective in diminishing anatomical parameters. The same observations were taken after treatment of diseased leaves with fungicides (contaf, canbinax and contaf + canbinax), the analysis expressed that in case of solo application of contaf, midrib thickness (1798.83µm), vascular bundle area

(2006643µm2) and parenchyma cell area (36787.88 µm2) reduced to its actual size, whereas by application of canbinax, only the midrib thickness (1847.28µm) was reduced at some extent. However, the combination of contaf and canbinax indicated healing effects, where midrib thickness (1750.37µm) and abaxial epidermal thickness (44.81µm) showed their actual measurements, adaxial epidermal thickness (97.51µm) and vascular bundle area (2680131µm2) showed higher measurement values than the normal, while parenchyma cell area (25284.32µm2) showed reduced value than the normal measurement (Figure 8).

PCA analysis

Principal component analysis (PCA) among leaf anatomical traits of healthy, disease inoculated and fungicide treated leaves (Figure 7). In first association adaxial epidermal thickness (ADET) was closely associated with contaf (CF) and Canbinax (CX) treated leaves. Midrib thickness (MIT) and parenchyma cell area (PCA) were closely linked with two inoculation stages i.e., stage 1 (S1) and stage 2 (S2). While the vascular bundle area (VBA) showed close association with healthy (HT) and contaf + canbinax (CF + CX) treated leaves (Figure 9).

Discussion

Black spot caused by Diplocarpon rosae, is the most destructive disease of field grown roses, that results in severe yield losses by damaging the leaves and flowers. Various disease management strategies are being implemented to combat lethal plant pathogenic infections, among all, synthetic chemicals are the quickest, easily available and sudden reactive substances that directly affects the pathogens and provides a speedy control (Ahmad et al., 2020). In the present study, five fungicides including Fossil, Contaf, Defeater plus 50% WG, Canbinax 45%DP and Agrosporter were used to assess their antifungal acuity against Diplocarpon rosae in-vitro. The data interpreted that, all the investigated fungicides have potency to suppress the mycelial growth of targeted fungus, but Contaf was found prominent with least mycelial growth followed by other treatments. Contaf contains hexaconazole, which restricts mycelial growth by inhibiting sterol demethylation pathway (Aguin et al., 2006). The outcomes of the current study are supported by the findings of Mansha et al. (2021), who discussed the efficiency of fungicides at distinctive levels showed against D. rosae. Similarly, Rehman et al. (2012) disclosed the effectiveness of Topsin M against mycelial growth of D. rosae at different concentrations. Correspondingly, Matte et al. (2022) reported the efficacy of Chlorothalonil against Diplocarpon rosae. Later on, Contaf and Canbinax as the most effective fungicides under lab conditions, were further investigated for field experiment. Aftermaths of field trial revealed that the combination of Contaf + Canbinax exhibited least disease incidence followed by solo application of Contaf and Canbinax. The findings of contemporary study are in line with the study of Matte et al. (2022); Bowen and Roark (2001) and Wojdyla and Lys (2000), who discussed the efficient management of black spot disease of rose using different fungicides.

The contemporary study also contains anatomical analysis of healthy, infected and treated leaf samples to understand the effect of pathogen on cellular level. Observations stated that, adaxial and abaxial epidermal cells, parenchyma cells area, midrib thickness and vascular bundle area were normal in a healthy leaf as compared to diseased leaf sample, where due to fungal penetration, parenchyma cells became larger in size, vascular bundle area was significantly decreased, the abaxial epidermis ruptured and adaxial epidermal tissues were sclerified. The current outcomes were consistent with the findings of Palmer et al. (1978), who studied the microscopy of healthy and Diplocarpon rosae affected rose leaves and noted that the healthy leaf consists of five tissues: The upper epidermis, the lower epidermis, spongy mesophyll, Palisade mesophyll cells, and xylem parenchyma cells surrounding vascular tissues. While after conidia germination, hyphae penetrate the cuticle, epidermal, mesophyll and parenchymatous cells of the host leaf. By expanding the trial, effect of pathogen on different traits were studied with different infection stages, and found that at initial stage of symptom appearance only the abaxial epidermis was found to be ruptured with no or minute effect of other examined traits, the same has been discussed by Gachomo et al. (2006) that only cell wall starts to degrade at initial stage of fungal penetration. In the mid stage of infection, the fungus started to penetrate in parenchymatous tissues and resulted ruptured parenchyma. However, in 3rd stage of infection, parenchyma cell size increased more than normal and the leaf tissues were also sclerenchymatous in nature. It has been confirmed by the findings of Gachomo and Kotchoni (2007), who noticed that with germination of conidia, a germ tube emerges from each conidium which penetrates into the host cuticle through a penetration peg. Our findings are also reflected by the work of Gachomo et al. (2006), who reported the formation of intracellular mycelia in both mesophyll and epidermal cells of infected leaf.

In our study, we also observed healing effect of fungicides (Contaf and Canbinax) on investigated anatomical traits of infected rose leaf cells. It was explored that by applying contaf, the epidermal and mesophyll cells were started to be normalized in their measurements, while with canbinax parenchyma cell area became decreased to the normal size. Finally, the combination of contaf and canbinax revealed significant healing where almost all investigated parameters expressed healing to their normal positions. The observations of our study are sustained by Gachomo et al. (2009), who reported the efficacy of strobilurins and triazole fungicides and found potential response of destruction of mycelial structures in the subcuticular, inter-cuticular and epidermal layers by triazole fungicides.

Acknowledgement

Authors are thankful to the Plant-Pathogen Interaction Laboratory, Department of Plant Pathology, University of Agriculture, Faisalabad, Pakistan for providing the facility and space for conducting experiments.

Novelty Statement

Anatomical analysis of black spot of rose caused by Diplocarpon rosae revealed an adverse effect of pathogen on physiology of the plant. Results of the contemporary study may provide a valid base for researchers to authenticate the major alterations caused by pathogen at cellular level and validate the responsive reactions of the plants against pathogenic infections.

Author’s Contribution

SF executed the field research and wrote manuscript, MA and NAR conceived the idea and supervised the work, MA and FA reviewed and edited the manuscript, GAK Helped in statistical analysis of the data, whereas AW and MR helped in conducting experiments and writing the manuscript.

Generative AI and AI-assisted technology statement

The authors declare that no generative AI or AI-assisted technologies were used in the preparation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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