Characterization and Pathogenicity of Rhizoctonia solani Kühn Associated with Sheath Blight in Local Rice Varieties (Oryza sativa L.) of Special Region of Yogyakarta

In Indonesia, rice is used as the first strategic and priority food commodity in agricultural development. However, in practice, efforts to increase it still experience various obstacles, one of which is caused by the attack of pathogen Rhizoctonia solani . This study aimed to determine the characters of R. solani and observe its pathogenicity on some local rice varieties in the Special Region of Yogyakarta. Four isolates of R. solani were used, namely RS MS2, RS MKP2, RS UMB, and RS PN, as well as six varieties of local rice varieties, which were Menthik Susu, Cempo Merah, Cempo Hitam, Segreng, Menoreh, and Hitam Makaryo, and national rice varieties IR 64 and Ciherang. Morphological characterization was carried out based on the cultural characteristics of the colony, hyphae, and sclerotia. A pathogenicity test was carried out using the Detached Leaf Inoculation method. The result showed that R. solani characters vary from whitish-brown, yellowish-brown, and light brown colonies; moderate and fast growth rates; abundant-aerial, moderate-aerial, and slight-aerial growth patterns; 90° hyphae branching, narrowing of hyphae at the branching point, and presence of sclerotia. The pathogenicity test gave the results of all isolates having varying pathogenicity levels on each rice variety. Menthik Susu, Cempo Hitam, and Hitam Makaryo were resistant to R. solani. Cempo Merah and Segreng were considered as moderately resistant varieties, whereas Menoreh was categorized as moderately susceptible. Ciherang and IR64 were susceptible varieties to R. solani.


INTRODUCTION
Rice plants (Oryza sativa L.) are a member of the family Poaceae, a cultivated plant and a national strategy commodity, and is used as a staple food of one-third of the world's population, including Asia. In Indonesia, rice is used as the first strategic food commodity and a priority in agricultural development (Somantri, 2001). Most of the land in Indonesia is planted with rice paddy, both superior varieties and local varieties. Local rice varieties are varieties that have been grown by farmers for decades and selected by nature. Although the production is not as high as superior rice varieties, local rice has various advantages such as good adaptability to the environment, good and fragrant rice, and are resistant against pest and disease attacks. The Special Region of Yogyakarta has approximately 20 varieties of local rice that spread throughout the district and city (Munandar et al., 1996;Hidayat, 2002;Bahagiawati et al., 2005;Kristamtini et al., 2018).
Various varieties of rice can be infected by fungal diseases. Attack of pathogens that cause disease on rice plants can decrease rice production in terms of quality and quantity (Shadily, 1984). One of the diseases that infect rice is sheath blight disease. In Indonesia, sheath blight is a disease that is easy to find and grows more severe from season to season, especially in areas of intensive rice farming (Nuryanto, 2011). This disease develops with varying severity and is thought to be closely related to the intake of technologies applied in farming by farmers (Nuryanto, 2003). Early symptoms of blight disease are oval or round brown patches on the leaf fronds (Utama, 2015). Planting short-life rice varieties with many seeds showed to be not resistant to sheath blight disease. This is thought to be triggered by the environmental conditions surrounding the plants which are warmer and moister (Eizenga et al., 2002).
Sheath blight is a rice disease that is caused by the fungus Rhizoctonia solani Kühn. The R. solani species have a fairly high diversity in terms of colony morphology, biochemical and molecular markers, pathogenicity and aggressiveness (Ajayi-Oyetunde & Bradley, 2018). High variability is an obstacle in managing R. solani because it provides a great opportunity for the emergence of new strains that are able to survive well and have high pathogenicity (Ou, 1985). Some varieties of rice in Indonesia showed moderate resistances to sheath blight caused by R. solani such as Inpara 3, Ciherang, Mekongga, whereas Indragiri and Inpari 13 were moderately susceptible (Muslim et al., 2012). Exploring resistances of local rice varieties from Special Region of Yogyakarta against R. solani or sheath blight disease has not been done and is required as an effort to minimize the decrease in productivity of rice yields. One effort that can be done is to find resistant varieties of rice to sheath blight. This study aimed to determine the characters of R. solani and observe its pathogenicity on some local rice varieties from Special Region of Yogyakarta

Morphological Characterization of R. solani
The isolates R. solani used was obtained from rice plants that showed symptom of sheath blight disease from fields in Special Region of Yogyakarta. Four isolates used were RS MS2 (Sleman), RS MKP2 (Kulon Progo), RS UMB (Yogyakarta), RS PN (Gunung Kidul). The four isolates were maintained and grown on Potato Dextrose Agar (PDA). This research was conducted in the Plant Systematics Laboratory, Faculty of Biology, Universitas Gadjah Mada. The observed cultural characteristics included macroscopic and microscopic observation. Macroscopic characters were colony diameter, colony growth rate and pattern, colony color, and sclerotia formation, including sclerotia size and shape. The microscopic characters observed were hyphae branching, hyphae size, and microscopic characteristics of the formed sclerotia. Microscopic observations were made by preparing slide preparations using tryphan blue and safranin dyes. Semi permanent slides were observed under light microscopes and optilabs with an objects magnification of 400×. The results were obtained then compared to related references. The growth rate of R. solani were measured and categorized as fast (>20 mm/day), moderate (15-20 mm/day), and slow (<15 mm/day) based on colony growth reaching a size of 9 cm or meet petri after incubation 48 hours (fast), 72 hours (moderate), and 96 hours (slow) (Mishra et al., 2014).

Pathogenicity Test of R. solani on Local Rice Varieties from Special Region of Yogyakarta
Pathogenicity test was conducted using the Detached Leaf Inoculation method from Jia et al. (2013). This research uses a Completely Randomized Design (CRD) that consists of 8 treatments (rice varieties) with 4 repetitions (R. solani isolates), so there were 32 in total. The rice seeds of six local varieties, consisting of Menthik Susu (Special Region of Yogyakarta), Cempo Merah (Sleman), Cempo Hitam (Sleman), Segreng (Gunung Kidul), Menoreh (Kulon Progo), and Hitam Makaryo (Bantul), and two superior national rice varieties (IR46 and Ciherang) were used in this experiment, sterilized and sowed. Then selected seedlings that showed uniform growth were planted in pots. After the plant was four weeks old, the second youngest leaf of each variety of rice plants was taken and cut to a size of 16 cm. The pieces of leaves with three replication of each treatment were placed on a plastic tray (31×24 cm) with a sterile tissue base that has been moistened with sterile water before. The leaves were inoculated by attaching 7-day-old R. solani mycelia with a diameter of 0.5 cm on each middle part of the rice leaf surface. After that, the tray was tightly closed with plastic wrap and incubated for 72 hours. The length of lesion Nafisa et al.: Characterization and Pathogenicity of Rhizoctonia solani Kühn Associated with Sheath Blight 149 was measured at 72 hours after incubation. The standard for evaluating disease score was set as 0 = no infection, disease area 0%, 1 = infection restricts at sites of inoculation, disease area <10%, 3 = restricted infection occurs at the site and within 2 cm of the site of inoculation, disease area 10.1-30%, 5 = unrestricted infection within 4-5 cm of the total infected areas, disease area 30.1-50%, 7 = expanded infection is greater than 5 cm length of the inoculated area, disease area 50.1-70% and 9 = the complete destruction of infection is greater than 6 cm length of the inoculated, disease area >70% (Jia et al., 2013). Disease Score (DI) was calculated using the following formula: sum of disease score x number of leaves within that score divided by the total number of leaves within treatment (Hua et al., 2014 with slight modification). Measurements were taken on the length of the lesions that appear later to determine the final reaction of the disease that appears on each variety. The reaction of the disease was determined based on the length of the lesion as resistant (R, <3.0 cm), moderately resistant (MR, 3.0-5.0 cm), moderately susceptible (MS, 5.1-6.5 cm), dan susceptible (S, >6.5 cm) (Jia et al., 2013).
The data was analyzed using the SPSS 25.0 program with one-way anova variance analysis based on F-test. If the data differs significantly, then followed by Duncan Multiple Range Test (DMRT) at 5% level.

Morphological Characterization of R.solani
Morphological observations showed that the four isolates ( Moderate-aerial mycelium obscured the surface mycelium, but without touching the cover, slightaerial mycelium did not obscure the surface mycelium. Several studies of R. solani morphology (Sunder et al., 2003;Lal & Kandhari, 2009;Mishra et al., 2014;Debbarma & Dutta, 2015;Gopireddy et al., 2017) showed that three-day-old young colonies of R. solani tend to be white and will turn brown as the colonies age. It is also mentioned that the colonies of R. solani had various colors: light brown, yellowish-brown, whitish brown, brown, dark brown, very pale yellow. Microscopic observations (Figure 2, Table 2) showed that the four isolates had hyphae diameters ranging from 6.20 μm to 8.62 μm, hyphae branches form 90° angles, narrowing occured at branching points, formation of monilioid cells (RS MS2, RS UMB, RS PN) and the formation of sclerotia in RS MKP2 isolates. There were several studies that showed diameter measurement results of hyphae varied. Hyphae width of R. solani isolated from maize in Andhra Pradesh, India ranged between 5.02-7.98 μm (Gopireddy et al., 2017). Singh et al. (2014) found that the hyphae width of maize isolates ranged from 5.418 to 10.764 μm while from rice isolates ranged between 7.338 to 10.036 μm. Lal and Kandhari (2009) and Kuiry et al. (2014) reported that the hyphae width of isolates from rice ranged from 4.73 to 7.43 μm and from 2.25 to 13.08 μm. The width of the hyphae of R. solani are generally larger than binucleate Rhizoctonia hyphae, and both hyphae can be easily distinguished by their size (Kasiamdari et al., 2002). The sclerotia of RS MKP2 were found on both the upper surface and the lower surface of the colony with a deep dark brown color. The number of sclerotia in each culture varied considerably in the range of 40-60 sclerotia with the size was 1-3 mm, and the shape was globose (Figure 3). In general, sclerotia is not formed in the culture medium as it considered as the survival propagule of R. solani (Mishra et al., 2014), sclerotia is reported as early inoculum of sheath blight in the rice field (Groth & Bond, 2007). The characteristics of the pathogen that grows quickly and produce sclerotia may increase the chances of a pathogen to survive and infect host plants. High sclerotial aggregation, though hinder in quick dispersal, helps in increasing the chances of germination because of the enhanced surface area (Madhavi et al., 2015). Basu et al. (2004) stated that there was no correlation between the mycelial growth of an isolate and its virulences on hosts. It was the abundance and size of the sclerotia that determined the virulences of an isolate. In older cultures, R. solani form the formation of monilioid cells (Andersen & Rasmussen, 1996).

Pathogenicity Test of R. solani on Local Rice Varieties from Special Region of Yogyakarta
Observation of pathogenicity tests were conducted on leaves from local varieties of rice inoculated with RS MS2, RS MKP2, RS UMB, and RS PN isolates at 72 hours after inoculation. Symptoms that appeared on the inoculated leaves showed varying results on each variety. The symptoms observed were grayish-white to brown color oval     (Inagaki, 2001). Infections can reach all parts of the leaf and cause symptoms of blight that can extend to the flag leaf (Nuryanto, 2017). As shown in Table 3, the leaves of Menthik Susu, Ciherang, IR64, Cempo Merah, Cempo Hitam, Segreng, Menoreh, and Hitam Makaryo responded differently to the infection of four isolates of R. solani (RS MS2, RS MKP2, RS UMB and RS PN). For each plant species, a wide variation of symptom severity induced by different R. solani was observed. The only R. solani isolates that did not cause disease on leaves tested was RS UMB inoculated on Hitam Makaryo variety. R. solani most severely infected Ciherang and IR 64 with a Disease Index (DI) of 3.19, followed by Menoreh with a DI of 1.08-1.13 with moderately disease infection. Compared to other rice varieties, Hitam Makaryo appeared to be the most resistant to R. solani isolates.
The pathogenicity studies on local rice varieties have shown that the virulence of R. solani isolates has a wide variation. A similar finding was reported by Taheri et al. (2007) that different R. solani isolates showed different pathogenicity on the specific cultivar of rice Oryza sativa cv. Zenith as shown in our study on Menthik Susu, Cempo Merah, Segreng, Menorah and Hitam Makaryo varieties. The difference in the degree of pathogenicity of R. solani isolates tested on six local varieties rice and two superior national rice can be influenced by the color of the colony. Colony color that are more concentrated (not white) tends to contain more melanin in cell walls and is related to the pathogenic ability. Isolates of pathogenic fungi that did not produce melanin were less pathogenic than those that produce a lot of melanin (Budiarti et al., 2020). It can also be associated with the growth rate of each isolate where the growth rate was moderate in RS MS2 and RS UMB and fast in RS MKP2 and RS PN. The rapid growth rate in isolates would support the occurrence of infection in plants.
To our knowledge, the resistance of local rice varieties against sheath blight disease used in this study has never been reported, except for Segreng (Dinas Pertanian dan Ketahanan Pangan Daerah Istimewa Yogyakarta [DPKP DIY], 2021). Results on the resistance of local rice varieties from Special Region of Yogyakarta (Table 4) showed that in the laboratory experiment, varieties of Hitam Makaryo, Menthik Susu, and Cempo Hitam were categorized as resistant with the length of lesion between 0-2.97 cm. In the field experiments, Hitam Makaryo was classified as resistant to fall, long-lived 172 days after sowing (dos) with the number of productive tiller 17 stems, Menthik Susu was classified as resistant to fall, long-lived 130-140 dos with the number of productive tiller 17 stems, and Cempo Hitam was classified as resistant to fall, long-lived 120 dos with the number of productive tiller 15 stems (Suharsono, 2018;Balai Pengkajian Teknologi Pertanian Jawa Timur, 2019;Nurhidayah & Umbara, 2019). Cempo Merah and Segreng were categorized as moderately resistant with the length of lesion 3.9-4.7 cm. Cempo Merah was classified as resistant to fall, medium-aged 109 dos with the number of productive tiller 10 stems and Segreng was classified as medium to fall, short-lived 100 dos with the number of productive tiller 9 stems (Balai Pengkajian Teknologi Pertanian Yogyakarta, 2006;DPKP DIY, 2021). The resistance of local rice Segreng has also been reported as moderately resistant to R. solani (Rustam, 2011;DPKP DIY, 2021). Menoreh was categorized as moderately susceptible with lesion    (Rustam, 2011), but resistant to leaf blight bacteria and pest aphrodisiacs. The information on the pathogenicity of Rhizoctonia solani to local rice varieties of the Special Region of Yogyakarta could be beneficial and new information for farmers and society in general.
The disease severity of sheath blight was high on short-type rice varieties with a large number of productive tillering and lower in tall-type rice varieties with a small number of productive tillering (Nuryanto et al., 2014). A large number of tillering will affect the microclimate under the canopy of plants causing warmer and moister condition that are suitable for supporting disease development, such as blight caused by R. solani (Kardin et al., 1988). Irawati and Hartati (2011) research shows that local varieties have good resistance to R. solani attacks.

CONCLUSIONS
The morphological characteristics of four isolates of Rhizoctonia solani (RS MS2, RS MKP2, RS UMB, and RS PN) isolated from sheath blight disease of rice showed variety in the colony, growth rates, growth patterns and can be recognized by its 90° branching, the production of monilioid cell and sclerotia. The four isolates of R. solani showed different pathogenicity on each local rice variety. New findings of the resistance of local rice varieties in the Special Region of Yogyakarta were shown in Hitam Makaryo, Menthik Susu, and Cempo Hitam that were categorized as resistant; Cempo Merah and Segreng were categorized as moderately resistant, and Menoreh were moderately susceptible. The rice variety of IR 64 was moderately susceptible to susceptible, while Ciherang were susceptible to sheath blight. These findings can be used as a reference for farmers to select sheath blight resistant local rice varieties for improving rice production.