Legume Research

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Molecular Characterization of Sclerotinia sclerotiorum Sacc. Initiating White Mold Disease in French Bean (Phaseolus vulgaris L.) and its Biological Management

Ramesh Singh Yadav1, Gaurav Kumar Yadav1,*, Amit Kumar Yadav1, Prashant Mishra1, Manoj Kumar Yadav2, Ajay Kumar Mishra1
1Department of Plant Pathology, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut-250 110, Uttar Pradesh, India.
2Department of Agricultural Biotechnology, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut-250 110, Uttar Pradesh, India.
  • Submitted10-01-2022|

  • Accepted23-05-2022|

  • First Online 29-06-2022|

  • doi 10.18805/LR-4872

Background: White mold disease caused by S. sclerotiorum is a serious peril to the cultivation of french bean in India. The disease primarily spreads by spores and normally in forms of sclerotia, which can remain viable in soil for several years and under appropriate environmental conditions, germinate to form mycelium, leading to infectious hyphae or producing apothecia.

Methods: In the present study, antagonistic activity of locally isolated bio-inoculants including four fungi and three bacteria were evaluated against white mold disease of french bean. In vitro efficacy of bio-inoculants was tested by dual culture method against S. sclerotiorum

Result: The molecular characterization of isolated pathogen was performed using ITS sequencing. The sequence length of the pathogen was 516 bp and approximately identical to publicly available S. sclerotiorum sequences on NCBI data base. In vitro study showed that, P. fluorescens isolate Pf008 was best causing 95.55 per cent mycelium growth reduction. In field experiment, the bio-inoculants were tested by various inoculation methods. Sclerotinia sclerotiorum infested soil treated with bio-inoculants as seed treatment and soil application. Results revealed that, seed treatment with formulation of T. harzianum isolate TS004 was best and enhance the growth promotion and increase the grain yield i.e. 13.93 q/ha and 12.10 q/ha during the year 2018-19 and 2019-20 respectively. Soil application with formulation of T. viride isolate TS006 was the utmost effective in reducing disease incidence by 83.91% during the year 2018-19 and seed treatment with formulation of T. viride isolate TS006 reducing disease incidence by 74.83% in the year 2019-20.
French bean (Phaseolus vulgaris L.) is an important legume crop belongs to family Fabaceae. It is also known as common bean, green bean, dry bean, rajma in North India and. Among food legumes the french bean has been the third most important worldwide famous crop, superseded only by soybean and peanut. There are two major commercial classes of french bean varieties, snap and dry beans (Jhanavi et al., 2018). It is esteemed for its protein rich (23%) seeds. It is a cold weather crop and highly sensitive to frost, high temperature and excessive soil moisture. (Sabate et al., 2018). In India, the crop is grown mainly in the states of Maharashtra, Jammu and Kashmir, Himachal Pradesh, Uttar Pradesh, Tamil Nadu, Kerala, Karnataka and West Bengal.
               
French bean suffers from a number of fungal diseases. However, among the fungal diseases, white mold caused by the fungus Sclerotinia sclerotiorum Lib. is a very detrimental disease affecting the french bean in the province of India. The disease is also known as sclerotinia wilt or stalk rot. The fungus is favored by temperate climates, moderate temperatures and high relative humidity (Mamani Gonzales et al., 2015). S. sclerotiorum primarily spreads by spores and normally in forms of sclerotia, which may infect stems, leaves and blooms and even spread to neighboring plants (Zhou and Boland, 1998). Sclerotia of the pathogen can remain viable in soil for several years and under appropriate environmental conditions, germinate to form mycelium, leading to infectious hyphae or producing apothecia, which release millions of airborne ascospores (Bardin and Huang, 2001). One of the major problems in managing this disease, that the pathogen produces large numbers of sclerotia which could stay viable for a long time in the soil (Alsum et al., 2017). This fungus can cause devastating economic losses in the harvests; therefore, its management is of regional importance. As for the last strategy, intensive use of chemical substances in crop management has led to microbial pathogen resistance to fungicides and has also caused serious problems for human health and the quality of the environment (Sabate et al., 2018). Therefore, it is necessary to explore the alternative of chemical management of disease. The study’s goal was to assess the efficacy of some locally available bio-inoculants (fungi and bacteria) in managing white mold on French beans.
The in vitro experiments were conducted to manage white mold disease of french bean caused by Sclerotinia sclerotiorum with isolates of different bio-inoculants at Centre of Excellence for Sanitary and Phytosanitary (SPS), Department of Plant Pathology and Crop Research Centre (CRC) of Sardar Vallabhbhai Patel University of Agriculture and Technology, Modipuram, Meerut, U.P.
 
Isolation and purification of the pathogen
 
The isolation and purification of the S. sclerotiorum was done by using the methodology of Jia (2009). Fragments of hyphal growth from the growing tips were transferred to fresh PDA slants. Pure culture was made, following repeated hyphal tip transfer (Alsum et al., 2017).
 
Molecular characterization of the pathogen
 
Molecular characterization of the isolate pathogen involved partial amplification of the ITS region (Freeman et al., 2002), followed by nucleotide sequencing. For this, ITS1 and ITS4 primers were used, following the conditions and procedure reported by Rahman et al., (2015). Phylogenetic analysis of the generated sequences was carried out using the MEGA6 program, with the sequences of Sclerotinia sclerotiorum  showing the highest homologies in Gen Bank.
 
Collection of bio-inoculants
 
The isolates of different bio-inoculants (Table 1) were collected from the Centre of Excellence for Sanitary and Phytosanitary, Department of Plant Pathology, SVPUAT, Meerut, Uttar Pradesh (India).
 

Table 1: List of different bio-inoculants used against Sclerotinia sclerotiorum.


 
Screening of bio-inoculants against pathogen suppression in-vitro condition
 
Antagonistic activities of microorganisms were tested against the soil borne plant pathogen Sclerotinia sclerotiorum by employing dual culture techniques of Rahman et al., (2009) on PDA.
 
Test for seed germination by roll paper towel method
 
The seeds were treated with the formulation of each bio inoculant and placed on moist paper towels (50-100) at equal distance and covered with another moist paper towel and rolled carefully without disturbing the already arranged seeds. Tie the rolled paper towel with a rubber band at both the ends. To avoid water loss, rolled towels containing seeds was wrapped with polythene and incubated for four to five days at room temperature. Examined germinated and ungerminated seeds by naked eyes (Kesharwani et al., 2018).
 
Screening of bio-inoculants on growth promotion and white mold disease suppression of french bean under pot condition
 
On the basis of in vitro result the selected bio-inoculants were evaluated for growth promotion of french bean. The selected fungal bio-inoculants were mass multiplied on Bajra seed and bacterial bio-inoculants were multiplied in nutrient broth medium. The soil was treated with selected bio-inoculants (at the rate of 5 g/kg of soil for fungal and 50 ml/kg of soil for bacterial bio-inoculants) and seed were sown in 8 inch and 16 cm diameter plastic pot at 2 cm depth. Soot length and root length were recorded at 45 days and 90 days after sowing. 
 
 
 
Evaluation of formulated bio-inoculants against white mold diseases suppression and growth promotion in french bean under field condition
 
The bio-inoculants were evaluated for growth promotion and management of white mold disease of french bean under field conditions at Crop Research Centre, SVPUAT, Meerut, Uttar Pradesh. Seeds were sown on November, 2018-19 and 2019-20 during Rabi season. The experiment was carried out following RBD with three replications. Size of the plots was kept 4.0 m × 3.0 m and plant spacing was kept 45 × 10 cm for french bean.
               
Experimental plots were made sick by soil inoculating @ 50 g/m2 with inoculum of Sclerotinia sclerotiorum. Fifteen treatments (including one control) were evaluated using four Trichoderma spp. isolates i.e. TS001, TS004, TS006, TS007, two Pseudomonas fluorescence isolates i.e. Pf008, Pf024 and Bacillus spp. isolate B005 used as seed treatment and soil application with FYM. The seeds were treated with formulated bio-inoculants @ 10 g/kg of seed (2×106 cfu) of Trichoderma spp., Pseudomonas fluorescence (2×108 cfu) and Bacillus spp, (2 × 108 cfu) and 6 kg/ha for soil application with FYM. During the growing period the plots were inspected regularly to record the incidence of disease from seedling to maturity stage of the crop. Data were recorded on percent disease incidence, shoot length, root length, number of branches, number of pods/plant and yield. The per cent disease incidence was calculated by the following formula:

Molecular characterization of Sclerotinia sclerotiorum through ITS sequencing and phylogenetic analysis
 
The molecular characterization of an isolated pathogen was performed using ITS sequencing. The nucleotide sequence generated in this study was assigned GenBank accession number MW173486.1. The isolate (svpuat) was attributed to Sclerotinia sclerotiorum. The length of the ITS sequence of isolate was 516 bp and almost identical to publicly available Sclerotinia sclerotiorum sequences (Fig 1 and 2). The phylogenetic relationship among the isolate and the related species, including S. sclerotiorum (e.g., GenBank accession numbers MW342187, KT595416 and LC318707 etc.), is shown in Fig 1. The results revealed that the isolates belong to a similar group of the type strain. This observation was similar to previously reported descriptions of Rahman et al., (2020). The study of Ciesniewska et al., (2017) revealed that, internal transcribed spacer ITS1/ITS4 PCR primers are effective for rapidly and accurately differentiating between the two species of Sclerotinia, they resulted a 540-bp product with all isolates tested (24 S. sclerotiorum and 26 S. trifoliorum).
 

Fig 1: A phylogenetic tree generated using the neighbor-joining method showing the genetic relationship between the Sclerotinia sclerotiorum isolate svpuat and other Sclerotinia sclerotiorum isolates available in GenBank (NCBI).


 

Fig 2: Agarose gel electrophoresis.


 
Screening of bio-inoculants against pathogen suppression in vitro
 
A total of seven bio-inoculants tested against the Sclerotinia sclerotiorum. On the basis of their antagonistic efficiency against pathogen (Line Fig 1), it was observed that all isolates inhibit the mycelial growth of Sclerotinia sclerotiorum. The maximum percent inhibition of pathogen growth 95.55% was observed in Pseudomonas fluorescens isolate-Pf008 against Sclerotinia sclerotiorum (Fig 3 and 4). The study of DeMelo and Faull (2000) reported that, T. harzianum and T. koningii were effective in inhibiting the mycelium growth of R. solani. A Similar result was obtained by Dutta et al., (2008). They studied the in vitro efficacy of bio-inoculants by dual culture method against S. sclerotiorum. Results revealed that, the best mycelium growth reduction was found to be caused by Pseudomonas fluorescens, which caused 64.93% mycelium growth reduction, followed by Bacillus subtilis (62.86%) and Trichoderma harzianum (59.08%). However, Sharma et al., (2016) recorded maximum 69.8 % inhibition of S. sclerotiorum with T. viride and minimum by Bacillus subtilis (42.2%).
 

Fig 3: Efficacy of different bio-inoculants against Sclerotinia sclerotiorum.


 

Fig 4: Effect of different bio-inoculants on seed germination.


 
Seed germination test
 
The results showed that (Fig 5) Trichoderma harzianum isolate TS004 was best which showed 93.33% seed germination followed by formulation of Trichoderma koningi isolate TS007 with 90.00% seed germination. Similarly, Kumar et al., (2018) reported that maximum (85.82%) seed germination was observed in Trichoderma viride and minimum seed germination was found in Pseudomonas fluorescens (82.32%) as compared to control (70.96%). The study of Dubey et al., (2017) showed that seeds treated with Pusa 5SD (T. harzianum-IARI P4) in combination with P. fluorescens gave the highest (97.9%) seed germination.
 

Fig 5: Antagonistic activity of bio-inoculants against Sclerotinia sclorotiorum.


 
Effect of different inoculation techniques of bio-inoculants on growth promotion and suppression of white mold disease of french bean under pot condition
 
 The result form the Table 2 indicated that all the inoculation techniques of bio-inoculants were found effective in growth promotion and suppression of disease. The maximum germination (93.33%), growth promotion viz. shoot length (48.35 cm), root length (15.58 cm) and minimum disease incidence (7.06%) with reduction of disease (87.29%) over control was recorded in seed treatment with Trichoderma harzianum isolate TS001. While, in control germination percent (60.00%), growth of plant viz. shoot length (32.56 cm), root length (8.28 cm) and disease incidence (55.55%) was recorded. The study of Zhang et al., (2016); Elsheshtawi et al., (2017) and Sumida et al., (2018) evinced that fungal bio-inoculants, such as Trichoderma asperelloides, Trichoderma harzianum and Coniothyrium minitans have been considered to be a viable alternative for the treatment of soybean seed. The result was also concordance with the findings of Dubey et al., (2017) they, treated seeds with Pusa 5SD (T. harzianum-IARI P4) in combination with P. fluorescens gave the seed germination (97.9%), pod yield (26.7 g/pot) and lowest disease incidence (4.50%). Soil application of Pusa bio-pellets of T. harzianum (IARI P4) 4.5% and T. viride (IBSD T-20) 9.00% also reduced disease incidence.
 

Table 2: Effect of different inoculation techniques of bio-inoculants on growth promotion and suppression of white mold disease of french bean under pot condition.


 
Evaluation of formulated bio-inoculants against white mold disease suppression and growth promotion in french bean under field conditions
 
Growth promotion
 
All the bio-inoculants were found to enhance the growth promotion and increase the yield as compared to control (Table 3). Data recorded during the year 2018-19 showed that seed treatment with formulation of Trichoderma harzianum isolate TS004 was best followed by soil application with formulation of Trichoderma harzianum isolate TS004 for growth promotion and increased the yield of french bean. The maximum shoot length (48.46 cm), root length (18.40 cm), number of branches/plant (7.02), number of pod/plant (11.80), yield 13.93 q/ha and increase in the yield 69.25% over control was recorded in seed treatment with formulation of Trichoderma harzianum isolate TS004. While, in case of control shoot length (26.60 cm), root length (7.40 cm), number of branches/plant (3.26), number of pods/ plant (4.33) and yield (8.23 q/ha) were recorded.
 

Table 3: Effect of formulated bio-inoculants on growth promotion of french bean under field conditions during year 2018-19 and 2019-20.


       
Similarly, in the year 2019-20, data showed that, seed treatment with the formulation of Trichoderma harzianum isolate TS004 was best followed by seed treatment with the formulation of Trichoderma viride isolate TS006 for growth promotion and increased the yield of French bean. The maximum shoot length (46.56 cm), root length (15.23 cm), number of branches/plant (9.26), number of pods/plant (10.40), yield 12.10 q/ha and increase in yield 67.82% over control were recorded in seed treatment with the formulation of Trichoderma harzianum isolate TS004. However, in case of control shoot length (28.30 cm), root length (7.26 cm), number of branches/plant (2.86), number of pod/plant (3.86) and yield (7.21/ha) was recorded. The study of Dutta and Das, (1999) revealed that seed treatment with Trichoderma harzianum provided the highest yield (69.51 q/ha), followed by Bacillus subtilis and Pseudomonas fluorescens. The lowest yield (41.00 q/ha) was recorded in control plot where S. sclerotiorum was applied alone. Increased shoot length with higher yield observed in this study might be associated with a reduction in white mold incidence. Likewise, Dubey et al., (2017) reported that seeds treated with Pusa 5SD (T. harzianum-IARI P4) in combination with P. fluorescens gave the highest shoot (27.1 cm), root length (10 cm) and dry plant weight (1538.9 mg/plant). Soil application of Pusa bio-pellets of T. harzianum (IARI P4) and T. viride (IBSD T-20) also enhanced the shoot, respectively root lengths, dry plant weight.
               
Disease reduction
The bio-inoculants which were inoculated as seed treatment and soil application showed better results for reduction of disease incidence against white mold disease in french bean during 2018-19 (Table 4, Fig 6). The finding evinced that soil application with formulation of Trichoderma viride isolate TS006 was recorded best among all the treatments with minimum per cent disease incidence (8.59%) was recorded at 90 days after sowing with an reduction 83.91% of disease incidence over control in this treatment, followed by (8.69%) disease incidence reduction 83.72% of disease incidence over control was recorded in seed treatment with formulation of Trichoderma harzianum isolate TS004 at 90 days after sowing. However, disease incidence 53.40% was recorded in control (without treated).
 

Table 4: Effect of formulated bio-inoculants on suppression of white mold disease in french bean under field conditions during year 2018-19 and 2019-20.


 

Fig 6: A) Infected plant with Sclerotinia stem rot symptom observed in french bean. B) Sclerotia produced by Sclerotinia sclerotiorum on pod of french bean.



Similarly in the year 2019-20 the minimum per cent disease incidence (6.69%) was recorded at 90 days after sowing with reduction of disease incidence (74.83%) over control in the seed treatment with formulation of Trichoderma viride isolate TS006, followed by 7.26% percent disease incidence reduction of disease incidence (72.68%) over control was recorded in seed treatment with formulation of Trichoderma harzianum isolate TS004 at 90 days after sowing. While, 26.58% disease incidence was recorded in control (without treated). The study was concordance with the finding of Sharma et al., (2016) they reported that minimum disease intensity (26.0%) was recorded with seed + soil application of T. viride followed by soil application of T. viride (35.7%) as compared to control (68.3%).  Similarly, Kumar et al., (2018) evinced that the minimum pre-emergence (13.93%) and post-emergence (17.00%) root rot disease incidence was recorded in seed treatment with T. viride followed by seed treatment with T. harzianum. Maximum pre-emergence (26.04 per cent) and post- emergence (31.50 per cent) root rot incidence were recorded in control.
Harmless and ecological alternatives to fungicidal management are urgent need to face increasing demand for safe, sustainable and effective management plans to white mold disease of french beans, counting on biocontrol agents and other best disease management tactics. This study demonstrated that the bio-inoculants could be considered a better alternative against Sclerotinia sclerotiorum than the fungicides in vitro and in vivo. The study suggests that all tested bio-inoculants could be used against S. sclerotiorum for management of white mold disease. Bacterial bio inoculant Pseudomonas fluorescens isolate-Pf008 was found best during in vitro study. Instead, during the field study, fungal bio-inoculants, specifically Trichoderma viride isolate-TS006 and Trichoderma harzianum isolate-TS004, were found to be the most effective for promoting growth and managing white mold disease of french bean.
Authors are thankful to Uttar Pradesh Council of Science and Technology (UPCST), Lucknow for providing fund and special thanks to Director of Agriculture, U.P. and Chief Secretary, Govt. of U.P., for providing fund for the establishment of Centre of Excellence for Sanitary and Phytosanitary (SPS) under RKVY scheme.
None.

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