2.1 Disease survey, symptoms study, and sample collection

A survey was conducted for the newly observed cosmos disease in Bangabandhu Sheikh Mujibur Rahman Agricultural Unviersity (BSMRAU), Gazipur, and its vicinity areas from 27 January to 8 February 2018. Four hundred and sixty-four cosmos plants from 10 cosmos gardens were randomly surveyed for suspected disease symptoms. Cosmos plants were mostly flowering to the late growth stage. Disease incidence was assessed as the percentage of symptomatic plants compared to the total number of surveyed plants. Severity was calculated following a scale of 0–5, where 0 = no symptoms, 1 = up to 10%, 2 = 11%–35%, 3 = 36%–65%, 4 = 66%–90%, and 5 = 91%–100% of the foliage affected by the pathogen [15]. The sclerotia developed inside the diseased tissues were collected by splitting open the stems and assessed for morphological characteristics. Diseased samples from 10 spots were collected and transported to the laboratory.

2.2 Isolation and preservation of the pathogen

Following preliminary washing with tap water, stems were dried on sterile blotter paper and then cut into 1–2 cm pieces. Dark-colored sclerotia were also collected from the infected stem. Infected tissue pieces and sclerotia were surface-sterilized in 70% ethanol for 2 min, then rinsed in sterile distilled water three times and dried on blotter paper. Later, tissue pieces and sclerotia were plated in Petri dishes containing potato dextrose agar (PDA) and incubated at laboratory temperature (25 ± 2°C) in the dark. Emerging mycelial colonies were transferred to new PDA plates. Ten pure isolates were obtained by hyphal tip isolation and marked as Isolate CSP1 (Cosmos Sclerotinia Pathogen 1) to CSP10. Pure isolates were cultured on PDA slants and preserved at 4°C.

2.3 Cultural and morphological characterization of the pathogen

The pathogen was initially identified based on the emerging colony, and morphological characters and their similarities with the previous reference works in the same laboratory [9, 10, 14, 15]. The pure mycelial isolates were transferred onto PDA plates and cultured at 25 ± 2°C in the dark for a week to study the colony characteristics. Permanent slides were prepared from the colony and examined under a light microscope to observe the fungal structures. These plates continued to incubate for 15 days to study sclerotial characteristics.

2.4 In planta pathogenicity test

Since all isolates of suspected pathogen appeared identical in colony morphology, a pathogenicity test was conducted with three randomly selected isolates (CSP1, CSP3, and CSP8) on the cosmos variety C. sulfurous. Seeds were surface-sterilized in 70% ethanol solution as described above and sown in seed trays (25.4 × 12 cm) filled with sterile soil. After germination, 2-week-old seedlings were transferred to earthen pots (12 × 12 cm) filled with sterile soil and grown in a net house under natural light and temperature conditions. Five-day-old actively growing fungal plates were used for inoculation. Three 4-week-old plants were inoculated with each isolate separately, and three plants were included as a control. Plants were wounded at the stem bark with a sterile blade above 10 cm of the soil surface (one wound per seedling) and inoculated with a 5 mm PDA plug. The inoculated stems were sealed with moistened cheesecloth. The seedlings were then covered with polybags and incubated in a dark, humid chamber for 48 h at 22 ± 1°C. After incubation, seedlings were shifted to the net house again. Seedlings were monitored daily for 20 days until the disease was developed. The suspected pathogen was reisolated from symptomatic tissues in three independent experiments to satisfy Koch's postulates.

2.5 Characterization of apothecia and ascospores of the pathogen

The isolated fungus was investigated to be a phytopathogenic member of the Sclerotinia genus by its ability to produce discal apothecia and eight binucleate ascospores. Fifty sclerotia were collected from three culture plates (CSP1, CSP3, and CSP8) to induce and study the apothecia. All sclerotia were surface sterilized with 1% NaOCl for 2 min, rinsed with sterilized distilled water two times, and air-dried. Five 9 cm sterile Petri dishes were filled with wet sterile sand. Surface sterilized sclerotia were placed inside the sand (10 sclerotia/plates) and incubated at 4°C in the dark for 6 weeks. The Petri dishes having germinating sclerotia were transferred to another incubator at 20°C under scattered fluorescent irradiation (260 μmol/m²/s) until apothecial discs were developed [15]. The morphological features of apothecia and ascospores were examined under a light microscope (40× and 100×). Fifteen apothecia were closely studied for morphological traits.

2.6 Molecular identification of S. sclerotiorum isolate CSP1 and CSP8

On the basis of the pathogenicity test results, one highly pathogenic isolate (CSP1) and one less virulent strain (CSP8) were chosen for molecular identification. Total genomic DNA was extracted from the mycelia of S. sclerotiorum isolate CSP1 and CSP8 as described by Islam et al. [15]. Polymerase chain reaction (PCR) was conducted with forward primer ITS-1 (5ʹ-TCCGTAGGTGAACCTGCG G-3ʹ) and reverse primer ITS-4 (5ʹ-TCCTCCGCTTATT GATATGC-3ʹ) to amplify rDNA-ITS (Internal Transcribed Spacer) regions of the fungal isolates. Two complete sequences were obtained for each ITS region, and the BLAST search program was used to search for nucleotide sequence homology in GenBank. Highly homologous sequences were aligned using Clustal-X version 2.0.11 and manually adjusted as required. Neighbor-joining trees were generated using the MEGA X version. Bootstrap replication (1000 replications) was used as statistical support for the nodes in the phylogenetic trees. Based on maximum sequence homology percentage, query coverage, and the lowest E-value, ITS sequence data of four archived isolates of S. sclerotiorum retrieved from GenBank were selected for phylogenetic analysis. Additionally, sequences of three isolates of S. minor and S. trifoliorum were included as references, with Hypocrea lixii serving as an outgroup taxon (GenBank accession no. FJ 861393.1).

2.7 Mycelial growth and sclerotia formation of S. sclerotiorum at different temperature and pH regimes

To determine the optimum growing temperature and pH of the isolated pathogen, the effect of various temperature and pH regimes on mycelial growth and sclerotia formation of S. sclerotiorum isolate CSP1 (most virulent isolate was used) was evaluated in three independent trials. PDA media were prepared and inoculated at the center with a 5 mm diameter mycelium plug excised from a 5-day-old S. sclerotiorum culture. The culture plates were incubated at 5, 10, 15, 20, 25, 30, and 35°C. Likewise, the effect of pH on growth and sclerotia formation of S. sclerotiorum isolate CSP1 was evaluated by growing the fungus at acidic, neutral, and alkaline pH. PDA media were prepared in 9-cm-diameter Petri plates and adjusted their pH to 5.0, 7.0, and 9.0 using 0.1 N HCl and NaOH. Petri plates were inoculated from a 5-day-old S. sclerotiorum culture and incubated at 20°C in the dark. There were three replicates for each temperature and pH range separately, and each replicate consisted of three Petri plates. The mycelial growth of the fungus was measured at 24, 48, 72, and 96 h postincubation. The growth of the cultures was continued in the incubator for 15 days to obtain the maximum yield of sclerotia. After 15 days, the number and weight of sclerotia were measured.

2.8 Nutritional requirements for growth and sclerotia formation of S. sclerotiorum

2.9 Determination of host range of S. sclerotiorum isolate CSP1

By screening against the most virulent strain, the broader host range of the fungus was determined. Twenty-five seasonal and perennial horticultural plants which had not been reported as hosts of the pathogen previously from Bangladesh were inoculated with S. sclerotiorum isolate CSP1. Seeds of seasonal plants were collected from the Horticultural Research Center, Bangladesh Agricultural Research Institute, Gazipur, Bangladesh, and sown in plastic pots (12 × 12 cm) filled with sterile field soil. Five plants were grown for each genotype, allocating one plant per pot in a net house under natural light and temperature conditions. When plants were at 4-week-old, one fully expanded leaflet from each plant was cut and rinsed with sterile distilled water. The leaflets were dried between the paper towels and placed on water-soaked three-layered blotter papers in 9-cm-diameter Petri Dishes. For perennial plants, fully expanded, healthy-looking leaves were collected from the established plants in the Horticultural Garden of BSMRAU. Five leaves were prepared for each host in the same way as described for seasonal plants. A 3 mm mycelial plug of the mentioned 5-day-old isolate was placed on each leaf. The inoculated leaves were kept in the dark at 20°C for 48 h. Additional leaves were inoculated with autoclaved PDA plugs as controls. Then inoculated leaves were transferred to a growth chamber at a photoperiod of 14 h/10 h light/dark and 21°C. Five days after the pathogen challenge, disease severity was estimated by measuring the percent area diseased on the leaves.

2.10 Screening of cosmos genotypes for resistance against S. sclerotiorum isolate CSP1

A study was conducted to determine the resistance of the available cosmos genotypes in Bangladesh against the disease using a detached leaf assay. Seven genotypes were screened, four from C. sulphureus and three from C. bipinnatus. Seeds collected from the Department of Horticulture, BSMRAU, Gazipur, Bangladesh, were surface-sterilized in 1% NaOCl solution and sown in seed trays (25.4 × 12 cm) filled with sterile field soil. Two weeks after germination, seedlings were transferred to earthen pots (12 × 12 cm) filled with sterile field soil and grown for 4–5 weeks under natural light and temperature conditions in a net house. There were nine plants for each genotype. One fully expanded leaflet was cut from each plant, rinsed with sterile distilled water, dried between the paper towels, and placed on three-layered water-soaked blotter papers in 9-cm-diameter Petri Dishes. A mycelial plug of 3-mm diameter excised from the edge of the fresh culture of S. sclerotiorum CSP1 was placed on the center of the midrib of each leaflet. The inoculated leaves were kept in the dark at 20°C for 48 h. The same number of leaves inoculated with autoclaved PDA plugs served as controls. Then inoculated leaves were transferred to a growth chamber at a photoperiod of 14 h/10 h light/dark and 21°C. Five days after the pathogen challenge, disease severity was estimated by measuring the percent area diseased on the leaves. Based on disease severity, each genotype was further categorized as highly resistant (HR)—0.00%–0.99%, resistant (R)—1.00%–9.99%, moderately resistant (MR)—10.00%–19.99%, moderately susceptible (MS)—20.00%–29.99%, susceptible (S)—30.00%–39.99%, and highly susceptible (HS)— ≥40.00%.

2.11 In vitro chemical control of S. sclerotiorum CSP1

2.12 In planta chemical control of S. sclerotiorum CSP1

2.13 Experiment design and data analysis

The experimental design was completely randomized, consisting of three to five replications. The experiment was conducted at least three times. The statistics 10 program package was used for statistical analysis, and treatments were compared via analysis of variance ANOVA using the least significant difference test (LSD) at a 5% (p ≤ 0.05) probability level. Data were transformed using the arcsine transformation method when it was necessary.

3.1 Symptom study and detection of the disease

Out of 464 plants examined, 129 were recorded with the suspected disease symptom. In severely infected plants, leaves were dark brown and dried. Some of the infected plants were at the mature stage but yet to be senesced, whereas some of them died prematurely and showed a typical wilting symptom (Figure 1a). The disease incidence was 27.80%, with an average disease severity rating of 4.46. Both internal and external large brown to tan areas with necrotic tissues were observed in the infected stems (Figure 1b). White cottony mycelia and large black sclerotia were also detected along the stem pith when splitting the infected stem longitudinally. The sclerotia were round to irregular in shape and measured 3.0–9.8 × 1.0–6.0 mm in size (Figure 1b).

3.2 Isolation, morphological characterization, and preservation of the pathogen

On PDA plates, all 10 isolates produced identical mycelial colonies that were fast-growing with white aerial thin mycelia but relatively thick at the colony margin (Figure 2a). Globose to cylindrical sclerotia were produced at the growing margins of all colonies after 10 days, forming concentric rings and radiating lines (Figure 1a). The reverse colony showed a dark gray color (Figure 2b). The hyphae were hyaline, branched, and multinucleate under the microscope (Figure 2c). No conidia or conidiophores were observed. As is typical with sclerotia-producing fungus, there were three distinct phases of sclerotial development. The initiation stage occurred with whitish aggregation of mycelia at 5 days post culture (Figure 2d); the development stage turned the whitish aggregates into beige color at 7–10 days post culture (Figure 2e), and the maturation stage took place after 10 days of incubation when the sclerotia turned from white to dark color (Figure 2f). Sclerotia had a black outer rind and a white inner cortex and developed in various numbers, forms, and sizes. The number and weight of sclerotia produced per PDA plate ranged from 6 to 40 and 6.38 to 50.81 mg, respectively. The mean length and width of 50 sclerotia were 5.4 and 4.6 mm, respectively. Individual sclerotium measured up to 9 mm long and 6 mm wide. After 4 weeks of incubation, sclerotia germinated and gave rise to white gray stripes, forming receptacles and producing apothecia in 5–6 weeks (Figure 2g,h). A single sclerotium produced up to four apothecia. Initially, apothecia were white to gray, hollow with a thick rim (Figure 2g), and then progressively differentially into a saucer shape or a cup or funnel shape at full maturity (Figure 2h). The mature apothecia had a diameter of 4–6 mm and a stripe length of 8–19 mm (Figure 2h). A thin slice of apothecia was also observed under a microscope for detailed study. The hymenial layer was full of paraphyses and asci (Figure 2i). Asci were cylindrical with a tapered base. Each ascus had eight ascospores (Figure 2i). Ascospores were uniseriate, hyaline, one-celled, smooth, ellipsoid, and uniform in size (Figure 2i).

3.3 In planta pathogenicity tests

Pathogenicity tests were performed on three of the 10 isolates, CSP1, CSP3, and CSP8 of the suspected pathogen. In the pathogenicity assays, all three isolates caused an infection on inoculated plants. Isolate CSP1 appeared more virulent than CSP3 and CSP8, causing infection rapidly in a greater proportion. The disease started with water-soaked and light tan lesions around the inoculation point by 72–96 h postinoculation, depending on the isolates. Later the lesions progressed both up and downward in the stem and created a distinct necrotic lesion within 5–7 days of inoculation. Eleven to fifteen days after inoculation, all inoculated plants wilted and finally died. Whitish mycelium and prominent sclerotia of S. sclerotiorum were visible inside the infected and dead stems. Whitish mycelia and sclerotia also emerged from collapsed tissues when kept in vitro moist chambers in the laboratory. Mycelia were transferred to the PDA plates, and new colonies were produced, which appeared identical to those recovered from field-infected plants. No disease symptoms were recorded in noninoculated control plants.

3.4 Molecular identification of the pathogen

The length of ITS sequences of two isolates (CSP1 and CSP8) obtained in this study was 572 bp (Figure 3a). The homology search through the GenBank DNA database revealed a 100% sequence identity with ITS rDNA sequences of several S. sclerotiorum isolates. A phylogenetic tree constructed with isolates of S. sclerotiorum, S. minor, S. trifoliorum, and H. lixii showed that the fungal isolates under study grouped with strains of S. sclerotiorum with strong bootstrap support (Figure 3b). Thus, the candidate fungus was identified as strains of S. sclerotiorum. The nucleotide sequences were deposited to GenBank under accession no. OL477344 and OL477345. There was, however, no distinction between CSP1 and CSP8 as indicated by ITS sequences; hence, the virulent isolate CSP1 was utilized to further characterize fungal physiology, nutritional requirements, host range, disease resistance, and fungicidal control.

3.5 Effect of temperature and pH on mycelial growth and sclerotia formation of S. sclerotiorum

The mycelial growth of S. sclerotiorum CSP1 was significantly affected by the temperature change from 5°C to 35°C. At 5°C, the fungus showed very sparse growth, acquiring only 9 mm radial growth 96 h after incubation. When the temperature increased to 10°C and 15°C, mycelial growth occurred faster, showing two- and threefolds higher growth than 5°C, respectively, 96 h after incubation (Figure 4a). However, increasing the temperature to 20°C resulted in the highest mycelial growth, producing a 40 mm colony diameter 96 h after incubation. Furthermore, an increase in temperature to 25 and 30°C reduced the growth by nearly half (21 mm) and ninefolds, respectively. However, at 35°C, the fungus growth was stopped entirely, and no growth was recorded even 96 h after incubation (Figure 4a).

Additionally, the fungus produced sclerotia in a wide range of temperatures. However, sclerotia formation was significantly affected by incubation temperature. The maximum number of sclerotia (36/plate) was formed at 20°C followed by 15°C (33/plate) (Figure 4b). Similarly, the total sclerotial weight per plate (38 mg) was maximum at 20°C (Figure 4c). Temperatures higher or lower than 20°C decreased the sclerotial yield of the fungus, although the reduction was more noticeable at higher temperatures. For this reason, the total sclerotial number and weight sharply declined at 25°C, while no sclerotium was formed when the incubation temperature was at or above 30°C (Figure 4b,c). Yet, individual sclerotial weight was the highest in plates incubated at 5°C (12.8 mg), followed by those at 10°C (12 mg), 15°C (11.2 mg), 25°C (10.7 mg), and 20°C (10.5 mg) (Figure 4d).

The highest mycelial growth was observed at pH 5.0 (Figure 4e). The growth of mycelium declined when pH became higher or lower than 5.0. The maximum reduction in the growth of the pathogen was observed at pH 9.0, while the lowest reduction was at pH 7 (Figure 4e). Similarly, the sclerotia number (44.2) and sclerotial weight (53.8 mg) per plate were the highest at pH 5.0 followed by pH 3.0 (22, 24.4 mg), pH 7.0 (18, 22.4 mg), and pH 9.0 (17, 13.8 mg) (Figure 4f,g). On the other hand, the maximum individual sclerotial weight was observed at pH 7.0 (12.4 mg), indicating that the sclerotial size was greater at pH 7.0 than at pH 5.0 (Figure 4h).

3.6 Effect of different carbon and nitrogen sources

As it was observed 96 h after culture, the highest mycelial growth of S. sclerotiorum CSP1 was recorded in glucose and mannitol (40 mm), followed by sucrose (36.6 mm) and insoluble starch (37.7 mm) (Figure 5a). However, the sclerotial number (28) and sclerotial weight (0.39 g) were recorded highest in glucose media (Figure 5b,c), while the lowest sclerotial number (14) and weight (0.2 g) were observed in mannitol media (Figure 5b,c). Similarly, different nitrogen sources significantly affected the growth and sclerotia formation of the pathogen. Peptone, yeast extract, and casein were the best nitrogen sources for maximum mycelial growth (39.2 to 39.4 mm) of S. sclerotiorum CSP1 (Figure 5d). Tryptophan and albumin gave the lowest mycelial growth (7.6 mm) after 96 h of incubation (Figure 5d). The yeast extract recorded the highest number (43.8) and weight (0.43 g) of sclerotia (Figure 5e,f). The sclerotial formation was inhibited entirely in tyrosine, tryptophan, and albumin media (Figure 5e,f).

3.7 Effect of different vitamin and mineral sources

All the five tested vitamins positively affected mycelial growth and exerted a statistically similar effect on the radial growth of S. sclerotiorum CSP1 96 h after culture (Figure 6a). However, they differed significantly among themselves in their effects on sclerotia formation. A significantly highest number of sclerotia was produced in minimal media amended with ascorbic (35.0) acid, followed by biotin (31.4) (Figure 6b). On the other hand, the lowest number of sclerotia was produced in media amended with thiamine (28.0), which was statistically similar to those produced by Riboflavin (29.0) and nicotinic acid (29.0) (Figure 6b). Similarly, the highest sclerotial weight (0.4 g) was attained in media containing ascorbic acid, and it was statistically similar to those produced by media containing biotin (0.39 g) (Figure 6c). Sclerotial weight was statistically lower in media containing other vitamins (Figure 6c).

The present experimental results also revealed that significantly highest mycelial growth (40 mm) of S. sclerotiorum was estimated in media containing KH₂PO_4, MgSO₄, NaCl, and CaCl₂ 96 h after culture (Figure 6d). On the other hand, the mycelial growth of this pathogen was slightly inhibited by CuSO₄, ZnSO₄, and MnSO₄ (26.4 mm) (Figure 6d). The sclerotia formation was supported by all minerals, while the highest number of sclerotia was observed in media containing MgSO₄ (36.60) and KH₂PO₄ (36.20) (Figure 6e). Media containing CuSO₄, and ZnSO₄, produced the lowest number (17) of sclerotia (Figure 6e). The sclerotial weight was maximum (0.40 g) in media containing MgSO₄. However, the minimum sclerotial weight (0.20 g) was recorded in the CuSO₄ rich medium, showing twofolds less sclerotial weight than MgSO₄ (Figure 6f).

3.8 Determination of host range of S. sclerotiorum isolate CSP1

Regarding pathogenicity tests on various host plants, individual plant species demonstrated differences in susceptibility to infection by S. sclerotiorum isolate CSP1. As many as 20 plant species of 10 families: Calendula officinalisi, Chrysanthemum indicum (Asteraceae), Catharanthus roseus (Apocynaceae) Daucus carota (Apiaceae), Raphanus sativus, Brassica juncea (Brassicaceae), Punica granatum (Lythraceae), Solanum tuberosum, Solanum lycopersicum, Solanum melongena, Capsicum annum (Solanaceae), Lablab purpureus, Phaseolus vulgari, Lens culinaris, Vigna radiata, Vigna mungo (Fabaceae) Spinacia oleracea (Amaranthaceae), Ipomoea batatas, Ipomoea aquatica (Convolvulaceae), and Elaeocarpus serratus (Elaecarpaceae) were severely infected by the pathogen showing a considerable level of disease severity (21%–95%) (Table 2). Cestrum nocturnum (Solanaceae) and Clitoria ternatea (Fabaceae) were also infected by the isolate of the pathogen but developed only a low level of disease (4%–6%). On the other hand, no infection (0%) was caused by the pathogen on three plant species, and thereby, no disease (0.0%) was observed on Ixora coccinea, (Rubiaceae), Quisqualis indica (Combretaceae), and Nerium oleander (Apocynaceae) (Table 2).

3.9 Screening of cosmos genotypes against S. sclerotiorum isolate CSP1

Water-soaked and light tan lesions developed at the point of inoculation 48 h after pathogen inoculation. Chlorosis and necrosis expanded into the entire leaf area within 4 days of inoculation. Leaves from all seven genotypes collapsed within 7 days of inoculation, and black sclerotia were formed within 10 days. The disease severity ranged between 44.25% and 100%, where the lowest and highest were recorded in CS1 (C. sulphureus 1) and CB2 (C. bipinnatus 2), respectively. Although individual genotypes exhibited differences in susceptibility to infection by S. sclerotiorum isolate CSP1, all were categorized as highly susceptible based on the disease severity scale (Table 3).

3.10 In vitro chemical control of S. sclerotiorum CSP1

In vitro efficacy of three popular systemic (Autostin 50 WDG, Amistar Top, and Ridomil Gold MZ) and two nonsystemic (Dithane M-45 and Rovral) fungicides were evaluated against the pathogen. Among the tested five fungicides, Autostin 50 WDG and Rovral completely inhibit (100% inhibition) the mycelial growth of S. sclerotiorum CSP1 at 100, 50, and 10 ppm of PDA (Figure 7a,b). On the other hand, Dithane M-45 completely inhibited mycelial growth at 100 and 50 ppm concentrations (Figure 7a,b). Amister top and Ridomil gold were only effective at 100 ppm, showing 88.5% and 80.12% inhibition of mycelial growth, respectively (Figure 7a,b).

3.11 In planta chemical control of S. sclerotiorum CSP1

Although the disease severity in the control plant was 95.37%, no symptoms were observed in the Autostin 50 WDG, and Rovral-treated leaves 1 day after spray (Figure 8). Autostin 50 WDG continued to protect the cosmos plants till 7 and 15 days after application, and no disease was observed in the treated plants during these periods. It means that the residual effect of Autostin was persistent and protected plants for a longer duration. On the other hand, the residual effect of Rovral faded away to some extent with time, and the disease severity in the treated leaves was 14.25% and 19.45% at 7 and 15 days after application, respectively. This shows that Rovral can also provide significant protection against the disease 15 days after application, although the efficacy was slightly lower than Autostin (Figure 8).