Evaluation of Some Fungicides in Controlling Blast of Rice var. Kalijira in Mymensingh

Rajia Sultana, Fatema-Tuz-Zohura, Md. Atikur Rahman, Ahmed Khairul Hasan, M Bahadur Miah, Muhammed Ali Hossain 1Plant Microbe Interaction Laboratory, Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh 2Department of Agriculture, Bangabandhu Sheikh Mujibur Rahman Science & Technology University, Gopalgonj-8100, Bangladesh 3Department of Agronomy, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh


Introduction
Rice (Oryza sativa L.) is the main food for half of the population of the world with the second-highest worldwide production after maize, which is also native to Asia, East, and South Asia (Boumas, 1985). Rice is the staple cereal crop of Bangladesh which provides nearly one-sixth of the national income of Bangladesh, accounting for nearly 20 percent of gross domestic production (Khandakar et al., 2013;Sayeed and Yunus, 2018). In 2019 according to the Bangladesh government, 36.2 million tons of rice produced with a seven-millionton surplus where the domestic needs were 29.1 million tons (AsiaNews.it, 2018). United States Department of Agriculture (USDA) recently forecasted that Bangladesh may produce 36 million metric tons of rice while Indonesia 34.9 million metric tons, India 118 million metric tons, and China 149 million metric tons in 2020/21 period which will make Bangladesh the 3 rd highest rice produced country (USDA, 2020). Rice is reported to be attacked by more than 70 different diseases caused by various fungi, bacteria, viruses or nematodes (Zhang et al., 2009), wherein Bangladesh rice is subjected to attack by 32 diseases (Kabir et al. 2015). Among them, rice blast caused by Pyricularia oryzae (anamorph), a fearsome fungal disease in Bangladesh. It's not a new disease in Bangladesh but it has been seriously breakout in the Boro season (winter rice) again in Bangladesh in 2017 and 2018 in the southern, central and northern districts of Bangladesh. Also, after severe damage to crops caused by a flash flood in Haor areas of Bangladesh, the widespread blast attack on Boro paddy may lead to a sharp decline in production in the year 2017. It was around 10-15 percent of the Boro paddy lands have been damaged by the blast fungus in the year 2017 and 2018 The officials of Bangladesh Rice Research Institute (BRRI) assume that over 5,000 hectares of Boro cropland in southern, mid-northern and northern regions were damaged by the neck blast attack (BRRI, 2019). P. oryzae is a seed-borne pathogen and can over-winter within plant debris, rice stubbles and also survive with the alternate host (Hubert et al., 2015, Pak et al., 2017. Disease severity is very much influenced by environmental factors and climatic changes. This blast pathogen might attack any of the growth stages of the rice plant from seedling to the premature stage of the crop. But leaf blast, node blast and neck blast, three different symptoms are observed in the affected organ of the infected rice plant. Leaf blast is characterized by eye-shaped spots on the leaves, neck and node blast are by their certain kind of necrosis. Node infection includes infected nodes appearing black-brown and dry and often occurs in a banded pattern. This kind of infection often causes the culm to break. The neck blast infects the panicle causing failure of the seeds to fill or causing the entire panicle to fall over as it is rotted. Out of three symptoms, neck blast is more destructive (Srinivas et al., 2011). Proper cultural practices, application of chemical fungicides and the use of resistant cultivars are the most important strategies for the management of rice blast till to date (Georgopoulos and Ziogas, 1992;Bekele, 2018;Rao and Kumar, 2018;Rijal and Devkota, 2020). Though production costs, the chance of environmental pollution and health hazards are high in chemical fungicides, chemical control of blast disease has become very much popular all over the world. Both seed treatments and foliar sprays with fungicides had been practiced to minimize the losses due to blast (Chaudhary and Sah, 1998;Chaudhary, 1999;Balgude and Gaikwad, 2019). From the last decade, Trooper and Nativo fungicides belong to Tebuconazole + Trifloxistrobin and Tricyclazole groups were identified as most effeeective against blast but these are expensive fungicides for the growers in Bangladesh. So, keeping this in mind, the effectiveness of some selected fungicides available in the market with their effective dose and spray frequencies has been evaluated both in in vitro and field conditions to combat Pyricularia oryzae.

Materials and Methods
Two phased experiments named laboratory and field experiment was conducted to evaluate the efficacy of some selected fungicides and their effective doses on controlling rice blast disease incited by Pyricularia oryzae in the Plant-Microbe Interaction Laboratory (PMIL), Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh.

Laboratory experiment
Stock culture of an aggressive isolate BD 576 of P. oryzae was collected from Plant Pathology Division, Bangladesh Rice Research Institute (BRRI), Joydebpur, Gazipur, Bangladesh. Then the number of pure cultures of P. oryzae isolate BD 576 was developed by inoculating the Oat Meal Agar (OMA) plates with the stock culture of BD 576 isolate and the petridishes were incubated in an incubator (model: FOC-215i, VELF Scientifica, Italy) chamber at 25˚C to provide the conditions for the pathogen to grow up to 10 days. Again, the approximately standard amount of fungal materials was transferred from 10 days old culture to the center of fresh PDA plates employing a sterilized block cutter for culture and allow them to grow for 10 days at 25°C temperature in an incubator. Sequential culturing from fungal stock was done for 3-5 times to get a pure culture that was used for screening of some selected fungicide against P. oryzae. Sixteen different fungicides with four different concentrations of each were used in the in vitro inhibition test (Table 1). OMA plates supplemented with different fungicides were inoculated with a pure culture of P. oryzae. After 10 days, the radial mycelial growth of Pyricularia oryzae was recorded by measuring the average of two diameters. The growth inhibition percentage was calculated by using the following formula (Al-Burtamani et al., 2005): Where, I= Growth inhibition (%), C = Mean mycelial growth (radial) of the pathogen in the control plate, and T = Mean mycelial growth (radial) of the pathogen in fungicide treated plate. This in vitro experiment was performed using a completely randomized design (CRD) of three replicates for each treatment. All analyses carried out using SAS (University Edition version 3.71 basic edition) statistical package.

Field experiment
Field trials were conducted in a piece of suitable land belong to Plant Pathology Field Laboratory, Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh. The experimental field was well-drained, medium high land with silty-loam textured soil. Kalijira, one of the high yielding traditional aromatic Aman rice (monsoon rice) variety in Bangladesh was used for this field experiment which is susceptible to blast disease. In this experiment, 36 plots were used and each of the plot size was 1.5m × 1.5m. The experimental field was fertilized with chemical fertilizers as per the recommended dose of the Fertilizer Recommendation Guide (BARC, 2012). After preparation of the plots, thirty (30) days old seedlings were transplanted in the well-prepared puddle field plots. Intercultural operations were done for ensuring and maintaining the normal growth and development of rice plants. Weeding was done three times and plots were irrigated when necessary.
Based on in vitro result, the possibility of available fungicides to the farmers and according to some related literatures, five best-performed fungicides (Nativo 75 WG, Companion, Autostin 50 WDG, Folicur EW and Contaf 5 EC) from different chemical groups with two different field effective concentrations (0.1% & 0.2%) were used to know their effect on the disease incidence and severity of rice blast, and different yield contributing parameters of rice. Ten fungicidal treatments and one control were used in this field experiment (Table 2). In this study, fungicide solutions were sprayed in 30 plots twice starting from the late booting stage. The first spray was done at 45 days after transplanting (DAT) at the late tillering stage and the second spray was done at 55 DAT i.e. seven days after the first spray application (at heading stage) (Kabir et al., 2004). The rest of the six plots were maintained as the control for this experiment.
Each of the plots was investigated for recording the incidence and severity of rice blast diseases. Affected plants from each unit plot were selected for assessing the disease incidence and severity. Data were recorded in 2-time points namely 52 DAT (7 days after the first spray) and 59 DAT (7 days after the second spray) by observing the typical blast symptoms on the leaf and neck region of the infected plant. Percent disease incidence was estimated by using the formula of Rajput and Bartaria, 1995. Disease incidence(%) = No. of infected plants Total no. of plants 100 Besides, the disease severity of naturally infected rice plants in 36 plots was assessed by a disease severity scale according to Mackill and Bonman (1992). The disease severity was also measured at 52 and 59 DAT i.e. seven days after spray application. The crop was harvested plot-wise on 15 November 2018 when 80-85% of the grains have become straw-colored. Data were collected on different yield contributing parameters namely plant height, number of total tillers per hill, number of effective tillers per hill, number of non-effective tillers per hill, number of infected panicle per hill, number of non-infected panicle per hill and weight of 1000 grains. The experiment was conducted in RCBD with three replications. The data on different parameters were statistically analyzed using the Analysis of Variance (ANOVA) technique to find out the level of significance.
The treatment means were compared by Duncan's Multiple Range Test (DMRT) at 5% level of significance. The collected data were analyzed using SAS (University Edition version 3.71 basic edition) statistical package.

Confirmation and Development of a pure culture of Pyricularia oryzae
After transferring the stock culture of P. oryzae isolate BD 576 to OMA containing plates, colonies with white color margin along with a blackish center on top and black coloured back appeared. The fungus as P. oryzae was confirmed by observing morphological features especially pear-shaped conidia under the microscope (Figure 1). Then slides were prepared from these colonies. An approximately standard amount of fungal material was transferred to the center of a fresh OMA plate using a sterilized block cutter for the re-culture and then grown under optimum incubated conditions for 10 days.

Effect of fungicides on radial mycelial growth of Pyricularia oryzae
The effect of fifteen selected fungicides with four different concentrations was observed on the radial mycelial growth of P. oryzae in this study.  Figure 2).

Effect of fungicides on the disease incidence and severity of rice blast
In this field experiment, the effect of five selected fungicides (Nativo 75 WG, Companion, Autostin 50 WDG, Folicur EW 250 and Contaf 5EC) were evaluated for disease incidence and severity for rice blast in the field conditions on the basis of their efficacy in in vitro experiment and their availability. Selected fungicides were sprayed at two different concentrations (0.1 & 0.2%) at 45 DAT and 52 DAT and the disease incidence data were recorded at 52 DAT i.e. seven days after first spray application. The different chemical treatments had a significant influence on the percent disease incidence of rice blast at 52 DAT (Table 4). However, in this study, the lowest leaf blast incidence (11.02%) was found in T1 (Nativo 75 WG @ 0.1%) treated plots in case of 0.1% concentrations followed by T5 (Autostin 50 WDG @ 0.1%) treated plots and the highest percent leaf blast incidence (41.26%) was observed in control (T0) plots followed by T9 (Contaf 5EC @ 0.1%) treated plots at 52 DAT (Table 4). In the case of 0.2% concentrations, the lowest leaf blast incidence (4.31%) was found in T2 (Nativo 75 WG) treated plots followed by T6 (Autostin 50 WDG) treated plots and the highest percent leaf blast incidence (41.26%) was observed in control (T0) plots followed by T10 (Contaf 5EC) treated plots at 52 DAT.
On the other hand, the disease severity of rice blast was evaluated against different chemical fungicides in two different time points (@ 45 DAT and 52 DAT). It is visible that the percent disease severity of rice blast (leaf and neck blast) was increased gradually with the advancement of crop growth. In the present research work, the lowest leaf blast severity 12.4% & 12.6% at 52 and 59 DAT respectively were found in T1 (Nativo 75 WG @ 0.1%) treated plots in case of 0.1% concentrations followed by T5 (Autostin 50 WDG @ 0.1%) treated plots and the highest percent leaf blast severity i.e. 16.3% and 20.5% were observed in control (T0) plots at 52 and 59 DAT respectively followed by T9 (Contaf 5EC @ 0.1%) treated plots (Table 4). In case of 0.2% concentrations, the lowest leaf blast severity 11.53% & 12.00% at 52 and 59 DAT respectively were found in T2 (Nativo 75 WG @ 0.2%) treated plots followed by T6 (Autostin 50 WDG @ 0.2%) treated plots and the highest percent leaf blast severity i.e. 16.3% and 20.5% were observed in control (T0) plots at 52 and 59 DAT respectively followed by T10 (Contaf 5EC @ 0.2%) treated plots (Table 4).

Effect of foliar spray of fungicides on the yield contributing parameters of rice
The different yield contributing parameters such as plant height, the number of tiller per hill, the number of effective tiller per hill, number of infected panicles, number of non-infected panicles and thousand-grain weight showed significant differences in treated and control plots in a field experiment ( Table 5). The maximum plant height (40.73 cm) was recorded in T2 (Nativo 75 WG @ 0.2%) treated plots followed by T1 (Nativo 75 WG @ 0.1%) and T6 (Autostin 50 WDG @ 0.2%) treated plots (Table 5). On the other hand, the minimum plant height (37.13 cm) was found in control plots followed by T9 (Contaf 5 EC @ 0.1%) treated plots (Table 5).     The maximum number of total tillers (15.33), effective tillers (14.40), non-infected panicle (13.67) and infected panicle (0.00) per hill were recorded for the T2 (Nativo 75 WG @ 0.2%) treated plots followed by T1 (Nativo 75 WG @ 0.1%) and T6 Autostin 50 WDG @ 0.2%) treated plots (Table 5), whereas the minimum total tillers (12.20), effective tillers (11.36), non-infected panicle (6.03) and infected panicle (5.30) per hill were recorded for the Control plots (T0) followed byT9 (Contaf 5 EC @ 0.1%) treated plots. To determine the effect of five selected fungicides on the weight of 1000 seeds (gm) of rice were also evaluated in this study and this parameter showed significant differences under different treatments. However, the weight ranged from 9.42 to 13.75g. In the case of weight of thousand seed, T2 (Nativo 75 WG @ 0.2%) treated plants showed maximum weight (13.75g) followed by T1 (Nativo 75 WG @ 0.1%) and T6 (Autostin 50 WDG 0.2%) treated plants while the lowest (9.42g) weight of thousand seeds was observed belong to the control plots.

Discussion
According to the results of in vitro study, Trifloxistrobin 25% + Tebuconazol 50%, Tebuconazol, Hexaconazol, Mancozeb, Zineb 68%+ Hexaconazole l4% and Carbendazim containing fungicides were more effective in controlling P. oryzae compared to Tricyclazole, Metalaxyl, Zineb, Pyraclostrobin, Metiram, Copper oxychloride containing fungicides. These findings are in agreement with the findings of Surapu et al. (2017) who stated that in in vitro conditions two fungicides namely Carbendazim showed complete inhibition (100% inhibition) of mycelial growth of P. oryzae at 1000 and 1200 ppm concentrations, in case of Tricyclazole complete inhibitions was recorded at 800 ppm and maximum growth inhibition was at 600 ppm. Chander et al. (2013) screened some fungicides under in vitro conditions each at a concentration of 0.1, 1, 10, 25, 50 and 100 ppm. Among tested fungicides, Tilt, Amistar top, Score and Folicur were found significantly effective over other treatments. Tilt exhibited 100% growth inhibition at 10 ppm while Folicur, Amistar top and Score exhibited 100% growth inhibition at 25 ppm. These studies revealed that Tilt followed by Amistar top, Score and Folicur are the most promising fungicides. Kunova et al. (2013) found in an in vitro study that the mycelium growth of Magnaporthe oryzae was inhibited to low concentrations of azoxystrobin and relatively high concentrations of tricyclazole. In the present study, the high concentration (0.1 & 0.2%) of tricyclazole (Tropper) showed better growth inhibition compared to low concentration (0.05%) in in vitro.

Conclusions
This research work was conducted to find out the efficacy of some selected fungicides against rice blast disease control in in vitro and field conditions. Based on the findings of the present study it may be concluded that some fungicides were more effective to inhibit Pyricularia oryzae at a very low concentration in in vitro condition as it inhibited radial mycelial growth up to 100% at only 0.0125% concentrations. On the other hand, Nativo 75 WG (Trifloxistrobin 25% + Tebuconazol 50%) @ 0.2% was found most effective for controlling blast of rice (leaf and neck), as the lowest percentage of leaf blast incidence (4.31%), lowest neck blast incidence (0.00%) and the lowest leaf blast severity 11.53% & 12.00% at 52 and 59 DAT respectively were found in Nativo 75 WG (@ 0.2%) treated plots followed by Autostin 50 WDG @ 0.2%. Plots sprayed with Nativo 75 WG @ 0.2% showed the best performance for yield contributing parameters like plant height (40.73cm), the number of tiller per hill (15.33), the number of effective tiller per hill (14.40), No. of infected panicle (0.00), number of non-infected panicles (13.67) and thousandgrain weights (13.75) showed significant differences in treated and control plots followed by Autostin 50 WDG @ 0.2%. However, two times the application of Nativo 75 WG or Autostin 50 WDG @ 2g/L of water starting from just panicle initiation will be effective for the control blast disease in field conditions.