MATERIALS AND METHODS

Fungal isolates: The causal fungus was isolated from the sclerotia that formed on the bulb of the infected amaryllis from San Kamphaeng District in Chiang Mai, Thailand in 2015. The sclerotia that had formed on the bulb of the amaryllis were disinfected with a 1% sodium hypochlorite solution for 3 min and rinsed with sterilized water two times. The surface-sterilized sclerotia were placed on Potato Dextrose Agar (PDA) and incubated at room temperature (28±4°C) for 4 days. The hyphal tips of fresh mycelia growing from sclerotia were cut and transferred to new PDA plates and incubated at room temperature. Morphological characteristic such as: mycelium condition and color, presence of hyphal clamp connection and fungal growth were noted¹².

Pathogenicity test and determining yield losses: The pathogenicity of the fungus was tested in the greenhouse on ‘Apple blossom’ and ‘Christmas gift’ amaryllis in pots. The test plants were inoculated with the isolated fungus using the following procedures. The fungal inoculum was produced in 200 g of sorghum in a plastic bag which was autoclaved at 121°C for 30 min. The mycelia from 10 discs were mixed with the sorghum and incubated at room temperature for 7 days. The sorghum infested with the fungal mycelia was thoroughly mixed with 3 kg of soil which were used to fill 30 cm diameter pots. One amaryllis bulb was placed in each pot and a CRD design with four replications was used. The ‘Apple blossom’ and ‘Christmas gift’ amaryllis without the pathogen served as controls, plant were grown in greenhouse and watered as required. The experiment ended after 6 months and the bulbs were lifted and their roots removed. All bulblets were counted and then removed. Bulbs were weighed and their circumferences measured.

Effect of temperature on radial growth: A culture of the fungal isolate from infected amaryllis was used. The linear hyphal extension rate (mm day^–1) of the fungal isolate was determined between 5 and 35°C on PDA in 9 cm diameter Petri plates. A sterile cork-borer (5 mm diameter) was used to remove an agar disc containing hyphae from the edge of an actively growing culture. The experimental design was a CRD with 5 replications. The daily radial fungal growth rate (mm day^–1) was calculated.

PCR amplification
DNA extraction: Fungal isolates were cultured in Potato Dextrose Broth (PDB) at room temperature (30±2°C) for 7 days. DNA was extracted from the isolate using a modification of the protocol of Stewart and Via¹². About 0.5 g of mycelia of the fungal isolate were ground with 12 μL of 2 mercaptoethanol and 2.4 mL of DNA extraction buffer (2% w/v CTAB, 1.42 M NaCl, 20 mM EDTA, 2% w/v polyvinylpyrrolidone, 5 mM citric acid and 100 mM Tris-HCl pH 8.0).

Table 1:	Active ingredients and chemical groups of the fungicides15
*NS: Non-systemic fungicide, S: Systemic fungicide, WP: Wettable powder, EC: Emulsifiable concentrate

Approximately 500 μL of the extraction solution were removed into a sterile 1.5 mL tube. Then 500 μL of a chloroform and isoamyl alcohol (24:1) solution were added and mixed. The solution was centrifuged at room temperature at 5000 rpm for 5 min. The upper aqueous phase containing the DNA was removed into a new sterile 1.5 mL tube. The genomic DNA was precipitated using 0.7X isopropanol by centrifugation at room temperature at 14,000 rpm for 20 min. Genomic DNA in TE buffer was visualized in a 1% (w/v) agarose gel after ethidium bromide staining. The DNA concentration was determined by gel electrophoresis¹³.

PCR amplification: The DNA amplification was performed by Polymerase Chain Reaction (PCR). The specific primer pair for detection of S. rolfsii included the SCR-F primer (5'-CGTAGGTGAACCTGCGGA-3') coupled with the SCR-R primer (‘5-CATACAAGCTA GAATCCC-3'), used to amplify a 540 bp product¹³. Sclerotium rolfsii specific PCR reactions were performed in a total volume of 25 μL, containing 50 ng of genomic DNA, 10X of PCR buffer, 10 mmol L^–1 of each dNTP, 50 mmol L^–1 MgCl₂, 1 U of Taq DNA polymerase and 0.5 mmol L^–1 of each primer. The reaction mixtures were incubated in a Peltier-based Thermal Cycler A100/A200 (LongGene Document Version 1.4). Following an initial denaturation at 94°C for 2 min, the DNA templates were amplified for 35 cycles consisting of 30 sec at 94°C,1 min at 58°C and 1.5 min at 72°C followed by a final extension at 72°C for 8 min. Amplification products were separated in 1% (w/v) agarose gels electrophoresis and viewed under UV light¹⁴.

In vitro fungicide bioassay: The isolate of Sclerotium sp. was maintained on PDA slant tubes at room temperature. For the in vitro fungicide bioassay, 5 mm diameter mycelial plugs taken from the edges of actively growing PDA cultures were inverted in the centers of PDA plates amended with fungicides to determine the effect of each fungicide on mycelial growth. Fungicides viz., captan, copper oxychloride, mancozeb, carbendazim and etridiazole+quintozene suspended in sterile distilled water were added to cooled, autoclaved PDA to give the desired concentration of the recommended dose, a ½ dose and a ¾ dose. The PDA without fungicide served as the control. Active ingredients and chemical groups of the fungicides are given in Table 1. There were five replicates of each treatment arranged in a CRD. The percent inhibition of mycelial growth was determined.

Statistical analysis: The study was conducted using a Complete Randomized Design (CRD). The data were subjected to statistical analysis using the Least Significant Different (LSD), the analysis of variance showed significance at an alpha level of 0.05.

RESULTS

Fungal isolates: The occurrence of disease in the bulb and leaves of ‘Apple blossom’ amaryllis was observed in the field. The fungus infects the surface of bulbs resulting in development of lesions. Numerous small white and brown sclerotial growths were observed on amaryllis bulbs, as well as blight along the leaf sheaths (Fig. 1a-b). The sclerotia were nearly round, 1-2 mm in diameter and initially white turning brown (Fig. 1c). The hyphae are 3.75-8.50 μm diameter. Primary hyphae showed clamp connections at the septa (Fig. 1d).

Pathogenicity test and determining yield losses: The pathogenicity test was conducted on ‘Christmas gift’ and ‘Apple blossom’ amaryllis in the greenhouse. White mycelia has started to form on the bulbs with sclerotia at 7 days after pathogen inoculation. After that, the sclerotial primordia developed into brownish sclerotia. Bulb rot symptoms were observed after the mycelium covered the bulb. Yield losses were caused by S. rolfsii in amaryllis 6 months after pathogen inoculation. The symptoms observed included blight along the leaf sheaths. Bulb fresh weight and circumference and number of bulblets were also significantly decreased by S. rolfsii infection compared to the non-treated control (Fig. 2, 3).

Fig. 1(a-d):	Bulb rot and leaf blight of amaryllis caused by Sclerotium rolfsii (a, b) Symptoms on leaves and bulb of amaryllis, (c) Mycelium growth and sclerotium on potato dextrose agar and (d) Clamp connection formed on the hyphae Scale bar = 10 μm

Fig. 2(a-c):	Effect of S. rolfsii on bulb quality of ‘Christmas gift’ amaryllis plants at 6 months after pathogen inoculation (a) Bulb fresh weight, (b) Bulb circumference and (c) Number of bulblets per plant

Effect of temperature on radial growth: Temperature significantly affected the linear hyphal extension rate of S. rolfsii. Fungal colonies grew at 10-35°C with a maximum extension rate of 18.1 mm day^–1 at 30°C. The isolate of S. rolfsii from amaryllis failed to grow at 5°C. The temperatures favouring the growth of the pathogen and infection of ‘Apple blossom’ amaryllis by S. rolfsii are 25-30°C.

PCR amplification: The isolate of Sclerotium sp. from ‘Apple blossom’ amaryllis was identified by using the SCR-F primer ('5-CGTAGGTGAACCTGCGGA-3') coupled with the primer SCR-R ('5-CATACAAGCTAGAATCCC-3'). This species-specific PCR confirmed that the isolate of Sclerotium sp. from ‘Apple blossom’ amaryllis was Sclerotium rolfsii, which was consistent with the morphological identification. A fragment of about a 540 bp was amplified from the genomic DNA of the S. rolfsii isolate (Fig. 4).

In vitro fungicide bioassay: Mycelium of S. rolfsii covered the petri plate surface after 48 h of incubation on unamended PDA.

Table 2:	Inhibition in growth of Sclerotium rolfsii on potato dextrose agar containing different concentrations of fungicides
*Values within a column followed by different letters are significantly different (p<0.05) by LSD. The experimental design was a Completely Randomized Design (CRD) NS: Non-systemic fungicide, S: Systemic fungicide

Fig. 3(a-c):	Effect of S. rolfsii on bulb quality of ‘Apple blossom’ amaryllis plants at 6 months after pathogen inoculation (a) Bulb fresh weight, (b) Bulb circumference and (c) Number of bulblets per plant

Fig. 4:	PCR amplification of a specific fragment from Sclerotium rolfsii
	Lane M: Molecular ladder, Lane 1: Distilled water, Lane 2: Sclerotium rolfsii from ‘Apple blossom’ amaryllis

Mycelial growth of S. rolfsii was markedly suppressed on media amended with etridiazole+quintozene, captan, mancozeb and copper oxychloride (Table 2). Increased fungicide concentrations resulted in corresponding reductions in mycelial growth. Etridiazole+quintozene was found to be the most effective treatment and gave 100% reduction in the growth of S. rolfsii when used at the recommended and ½ doses. At ¼ of the recommended dose a 91.89% reduction of mycelial growth was achieved. Captan and mancozeb at the recommended doses were found to be effective in inhibiting the radial growth of S. rolfsii by 68.85 and 64.56%, respectively. Carbendazim showed the lowest inhibition (4.30%) of mycelial growth at the recommended dose compared to the other fungicides tested.

DISCUSSION

A bulb rot and sheath blight disease on ‘Apple blossom’ amaryllis was recently noticed causing major crop loss in some commercial fields in San Kamphaeng District in Chiang Mai, Thailand. The initial symptoms were water-soaked lesions in bulbs followed by total bulb rot. White cottony mycelia formed on the bulb and blight occurred along the leaf sheaths, on which small white and brown sclerotia formed. The hyphal diameter was approximately from 3.75 to 8.50 μm. Therefore, the isolate was identified as Sclerotium rolfsii and confirmed by a species-specific PCR. The optimum temperature for growth rate (18.1 mm day^–1) was 30°C on PDA.

The pathogen causes significant crop damage worldwide. Similar observations were made by Kwon¹², who reported that S. rolfsii was found in garlic cultivated from 2008-2010, in Gyeongnam province, Korea. Early symptoms of this disease were water-soaked spots on bulbs, which were followed by rot, wilt, blight and death. White mycelia spread over the lesions near the soil and sclerotia formed on the stem. Hyphal diameter ranged from 4-8 μm. Sclerotia were round, tan to brown and ranged from 1-3 mm in diameter. Han et al.⁸ reported that a stem rot disease found in garlic and Cymbidium orchids in Korea, was caused by S. rolfsii (Saccardo). Jeeva et al.¹⁴ identified S. rolfsii by PCR analyses using specific primers. A species specific PCR performed by using two primers: SCR-F and SCR-R, amplified a 540 bp product from S. rolfsii DNA isolated from the plant Amorphophallus paeoniifolius.

The current research results showed that the fungicide combination of etridiazole+quintozene was the most effective and gave a 100% reduction in the radial growth of S. rolfsii on PDA when used at the recommended and half-doses. Captan and mancozeb were found to be the effective in inhibiting the radial growth of S. rolfsii at the recommended doses, producing growth inhibitions of 68.85 and 64.56%, respectively. Keyser and Ferreira¹⁶ found that quintozene as a dip and soil drench, effectively protected plants against S. rolfsii. Using quintozene before planting in the nursery can be a practical way of controlling the disease where S. rolfsii is a problem. Yaqub and Shahzad¹⁷ reported that at 100 ppm Dithane M-45 (mancozeb) showed a 60% inhibition in mycelial growth of S. rolfsii. Similarly, Dithane M-45 at 200 ppm reduced the growth of S. rolfsii¹⁸. Captan, chlorothalonil, mancozeb and pentachloronitrobenzene in agar reduced germination of sclerotia by 80-95%¹⁹. Mancozeb at the highest concentration of 400 ppm produced an 80.36% radial growth inhibition of S. rolfsii²⁰. Thiophanate methyl, metalaxyl+mancozeb and mancozeb at 50, 100 and 250 ppm concentrations reduced the mycelial growth of S. rolfsii ²¹. The importance of fungicide mixtures in reducing the probability of resistance/tolerance development in fungi has been discussed²². Moreover, carbendazim, captan and mancozeb inhibited the mycelial growth of the fungus at a 0.1% concentration by 54.3, 67.3 and 58.7%, respectively²³. Das et al.²⁴ reported that hexaconazole 5% EC and tebuconazole 25.9 m/m EC appeared to be the best in inhibiting the hyphal growth of S. rolfsii at a 50 ppm concentration while, captan and carbendazim at 100 ppm inhibited growth by 16.0 and 3.1%, respectively. This study showed that carbendazim produced the lowest inhibition (4.30%) of mycelial growth at a 500 ppm concentration. Manu et al.²⁵ found that carbendazim wasn’t inhibitory to the soil-borne pathogen at 250 ppm similar results were obtained by Rakholiya²⁶, carbendazim and copper oxychloride were found to be the least effective against S. rolfsii. Based on the results obtained from this study, we recommend application of etridiazole + quintozene to control S. rolfsii is recommended.

CONCLUSION

Bulb rot disease caused by S. rolfsii has become a serious and recurrent problem in amaryllis production areas of Chiang Mai, Thailand. The pathogen was identified by PCR analyses using specific primers and morphological characteristics. The current research results showed that the most effective treatment against the pathogen was the combination of the systemic fungicides etridiazole +quintozene, which inhibited the fungus in vitro by 100% at the recommended and a half-dose. The non-systemic fungicides captan and mancozeb were of intermediate effectiveness. Therefore, protection of amaryllis against S. rolfsii, may be achieved by treating the bulbs with captan or mancozeb before planting in the plantation field. Etridiazole+quintozene added to soil infested by S. rolfsii may also protect amaryllis from infection.

SIGNIFICANCE STATEMENT

In this study a disease of amaryllis caused by Sclerotium rolfsii was identified. The study showed that, etridiazole+quintozene at a 270 ppm concentration (half recommended dose) was 100% inhibitory to the radial growth of S. rolfsii in vitro.

ACKNOWLEDGMENTS

This research was supported by the Amaryllis Breeding Project and was carried out with the cooperation of Kebol B.V. (the Netherlands), Chiang Mai University (Thailand) and H.M. The King’s initiative Centre for Flower and Fruit Propagation (Thailand).