Evaluation of Some Fungicides against Collar Rot Disease of Soybean

A number of selected fungicides were evaluated to determine their efficacy for controlling collar rot disease of soybean plants caused by Sclerotium rolfsii. The experiment was conducted under the controlled condition at the Plant Pathology Laboratory and Field laboratory of BINA, Bangladesh Agricultural University campus from November 2018 to August, 2019. In-vitro research was done for the observation of radial mycelial growth of S. rolfsii on potato dextrose agar (PDA), treated with five fungicides viz. Antracol 70 WP (T1), Ridomil Gold MZ 68 WP (T2), Secure 600 WG (T3), Bavistin DF (T4), Dithane M-45 (T5) and one non-treated (T0) treatment. The highest percentage of mycelial growth inhibition of S. rolfsii in PDA medium was recorded in treatment T5 (Dithane M-45) 100% and lowest in treatment T3 (Secure 600 WG) 37.33% at 6 days after inoculation. Then the selected five fungicides were again applied to pot under controlled conditions to observe the best effect of selected fungicides against collar rot pathogen of soybean plants. The inoculation was done on a variety of BINA soybean 4 in pot condition. The highest mortality percent for the collar rot disease was found in treatment T0 (controlled) 100% soybean plants conversely, the lowest mortality percent was found in treatment T5 (Dithane M-45) 27.28% besides 38.92% in T2 (Ridomil Gold MZ 68 WP), 43.42% in T1 (Antracol 70 WP), 46.18% in T3 (Secure 600 WG) and 50.00% in treatment T4 (Bavistin DF) respectively. Thus, Dithane M-45 was found superior in controlling collar rot pathogen S. rolfsii of Soybean over all other fungicides tested in both in vitro and in vivo.


INTRODUCTION:
Soybean (Glycine max L. Merr.) is a leguminous crop and a good source of protein and vegetable oil. Soybean is providing 40% protein. It can play a vital role in balancing the protein deficiency of our diet (Mondal and Wahhab, 2001;Rahman, 2003). Soybean production of Bangladesh is 1.59 t/ha which is much lower than the world average (2.53 t/ha) (FAO, 2013). Soybean raw materials are used in different industries. Different soya foods are also prepared from soybean (Hossain et al., 1992).
Soybean can fix to an extent of 300kg Nitrogen/ ha/year and reduce the nitrogen requirement by 25-75% with Bradyrhizobium japonicum. Moreover, Soybean can fix atmospheric nitrogen symbiotically and therefore, through symbiosis about 80-90% nitrogen demand could be supplied by soybean (Keyser and Li, 1992). Thus this crop enhance soil fertility and economizes crop production not only for themselves but also for the next cereal crops and other non-legume crops grown in rotation and thereby, minimizing the regular rate of nitrogen fertilizer.
The production of soybean plants is low due to disease constrains. Soybean plants suffer from many diseases for example anthracnose, mosaic, collar rot, powdery mildew, etc. Out of them collar rot/ Foot and root rot of soybean is very severe damaging disease in Bangladesh, which is caused by S. rolfsii. It is common where high temperatures exist during the rainy season. The pathogen causes pre emergence and post emergence damping off, foot and root rot, collar rot and wilt of seedlings. Collar rot can occur in both seedling and adult plants. Collar rot /Foot and root rot disease is more common than southern blight or stem rot form in our country. Southern blight form of the diseases usually occurs on the older plants (Ahmed and Hossain, 1985). S. rolfsii, has the wide host range of more than 500 species of plants including many important crops such as maize, wheat, gram, khesari, lentil, mashkalai, mungbean, sunflower, sesame, brinjal, bitter gourd, bottle gourd, cowpea, cucumber, okra, radish, tomato, radish, chilli, coriander, garlic, onion, apple, peanut, soybean, and potato ; also many woody ornamentals, herbaceous annuals and perennials including ageratum, aucuba, azalea, begonia, columbine, coneflower, forsythia, hydrangea, marigold pansy, petunia, viburnum, and zinnia (Punja, 1985;Mullen, 2001).
Sclerotium rolfsii is both seed borne and soil borne pathogen (Fakir et al., 1991). Collar rot causes yield loss of 10-25%, but under severe diseased conditions yield losses may ranges from 50 to 80% (Patil and Rane, 1982). It is very difficult to manage the pathogen because of its diverse nature of survival as large number of sclerotia produced and their ability to persist in the soil for several years. The fungus causes 25-50% loss through infection of seedlings. To control the fungus people usually use many chemicals. But the fungus S. rolfsii is a facultative saprophyte and can maintain continuity of its generation under adverse situation by the formation of sclerotia (Ahmed, 1980). Varietal resistance is another option for diseases management (Singh et al., 2002). But there is no soybean resistant variety against collar rot in Bangladesh. So, it is necessary to search out effective fungicides to control this devastating disease. That's why the present research was taken under consideration to select the effective fungicides in vitro (Laboratory experiment) and in vivo (Pot Experiment) against collar rot disease of soybean caused by S. rolfsii.

MATERIALS AND METHODS:
The experiments were conducted during 2018 to 2019 in CRD design with thrice replication.

In-vitro evaluation (Laboratory Experiment) of different fungicides against Sclerotium rolfsii:
Poisoned food technique was used for evaluating the fungicidal effects on the growth of collar rot pathogen S. rolfsii. Five different fungicides viz., Antracol 70 WP (Propineb), Bavistin DF (Carbendazim), Dithane M-45 (Mancozeb), Secure 600 WG (Mancozeb and Fluazinam), Ridomil Gold MZ 68 WP (Mancozeb and Metalaxyl) were included in the experiment. The fungicides solutions were prepared in accordance with the labeled doses in the fungicide formulation. For example, preparation of 0.2% Antracol solution, 2 g of Antracol was taken into 1000 ml of sterilized distilled water. Poison food technique with little modification was followed for the evaluation of selected fungicides against S. rolfsii. Preparation of PDA containing fungicides was done in two steps.
In first step, clean peeled slice potato tubers of 125 g were boiled in 500 ml of water. Water was sieved from boiled potato slices and 10 g agar and 10 g Dextrose were added to it. PDA suspension was prepared up to 1000 ml of sterilized media before pouring in petridishes for preparing the original concentration. For preparation of poison food media 0.2g fungicide was mixed with 99.8 ml PDA media (Nene and Thapliyal, 1982). Isolation of the S. rolfsii was done by following standard tissue planting method from infected soybean plants. The infected tissues of soybean plants showing typical collar rot symptoms planted on potato dextrose agar (PDA) medium. The petriplates were incubated at 28+2 0 C were observed periodically for growth of the fungus. Pure culture of S. rolfsii was maintained on PDA slants for further studies. The pathogen was identified based on its mycelial and sclerotial characteristics (Sekhar et al., 2017). The amended medium was then poured in sterilized Petri dish (90 mm dia.). A 5-7 mm disc of test fungus cut from the margins of ten days old cultures were placed centrally in each of the Petri dish and were incubated at 27°C. Mycelial growth colony diameter of S. rolfsii was measured at two days interval. It took 12 days to coverage of petridishes by mycelia. The percent mycelia growth inhibition (PI) of the fungus over control was calculated by using following formula (Vincent, 1927). PI = C-T/C × 100. Where, C= Growth of test pathogens in absence of fungicides (mm), T= Growth of test pathogens in presence of fungicides (mm). The diameter was measured with mm scale from the back side of the petridish taking it opposite to light.

In-vivo evaluation (Pot Experiment) of different fungicides against Sclerotium rolfsii:
The in vivo evaluation of different fungicides was conducted in controlled conditions in the Field laboratory during April, 2019 to October, 2019. The experimental field was medium high land and the soil was sandy loamy. The experimental site belongs to Brahmaputra Alluvial Tract, the PH was 7.5. The soil of the experiment was collected from BINA farm field and then sun-dried, ground and screened through 10 mesh sieve to remove large particles and debris. The soil was dripped with 2% formalin solution 200ml per cft soil and kept covered with polythene sheets for 2-3 days. Then the soil was uncovered and kept for 4 days to release the gas formalin. Collected BINA soybean 4 seeds (10 seeds) were taken on a wet blotter paper in a petridish and kept for 4-7 days. Then the germination percent was 100% calculated by the formula of The seeds were treated with provax 200 0.2% before sowing. The soil was poured into perforated plastic pots at the rate of 11.50 kg/pot for 6 treatments including control. The following fertilizers ( Table 1) were applied in each pot as per recommendation dose of the fertilizers Recommendation Guide (BARC, 2012) and total fertilizer were mixed uniformly and 5.22g/ pot were given two days before seed sowing. The pots were tagged according to treatments received. Five fungicides ( Table 2) were selected for pot experiment based on laboratory performance. Total six treatments were applied including control. There were 3 replications of each treatments and pots with fungicidal subsequent foliar spray.

Data Analysis -
The data obtained from this research work were statistically analyzed following MSTAT-C program and treatment means in ANOVA tables were compared by LSD (0.05%) as followed by Gomez and Gomez (1984).

RESULT AND DISCUSSION:
The results were compiled based on the inhibition of radial mycelial growth and mortality percent of soybean plants. The collar rot symptoms of soybean plants showed mycelium covering the plant stem near the soil surface. The pathogen produced cottony mycelium with small brown sclerotia on the infected plant parts revealed the collar rot disease symptoms. Later, affected plants/branches turned yellow or drooped while retaining their green color, followed by drying and turning straw colored. White mycelial strands appeared at the collar region and above, covering the base of the branches. Whitish, brownish, irregular shaped sclerotia mingled with mycelial strands on branches. The similar findings were described by Rekha et al. (2012), Kokub (2007), Prabhu and Patil (2004), Gupta and Sharma (2000), etc. The pathogen S. rolfsii was found whitish in colour in culture media and small mustard seed like sclerotia also found. The infected seedlings also showed cortical decay at the collar region and whitish mycelium was found extended on the soil surface. These findings were supported by Kulkarni et al. (1995), Aken and Dashiell, (1991), and Baruah et al. (1980).

In vitro evaluation (Lab experiment) -
Efficacy of different treatments on radial mycelial growth of S. rolfsii was shown in Table 3.      0   20   40   60   80   100   T0  T1  T2  T3  T4  T5  Inhibition Percent   Treatments   T0 T1 T2 T3 T4 T5  T  On the other hand, Sharma and Ghosh (2017) reported collar rot disease yielded 55-95 % seedlings mortality of chickpea under heavy rainfall and high soil temperature (25-30°C). From the findings of the present experiment it may be concluded that treatment Dithane M-45 was most effective under invitro condition as it has completely inhibited mycelial growth of S. rolfsii up to 100% and exhibited minimum mortality percent of Soybean plants at 0.2% concentration. From this experiment, it was observed that Dithane M-45 reduced the growth of S. rolfsii significantly in in-vitro and reduced collar rot disease incidence of soybean in vivo condition.

CONCLUSION:
From the findings of the present experiment it may be concluded that Dithane M-45 was found completely inhibited the mycelial growth of collar rot pathogen Sclerotium rolfsii in vitro and exhibited minimum mortality percent of Soybean plants at 0.2% concentration. Again Dithane M-45 also showed superior performance in reducing collar rot incidence of soybean in pot experiment.

ACKNOWLEDGEMENTS:
The authors hereby acknowledge the supports of Plant Pathology Division of Bangladesh Institute of Nuclear in Agriculture for giving us the opportunity to conduct this research work.

CONFLICT OF INTERESTS:
The author (s) declared no potential conflicts of the interest with respect to the research, authorship and/or publication of this article.