Effectiveness of Fungicides against Colletotrichum lindemuthianum Causing Anthracnose of Green Gram [Vigna radiata (L.) Wilczek]

Green gram [Vigna radiata (L.) Wilczek, Syn.: Phaseolus aureus Roxb., Phaseolus radiata L.] is India’s third most important pulse crop after Bengalgram and redgram. Green gram is grown not only for seed, but also for green manure and fodder. On a dry weight basis, green gram contains about 25 to 28 per cent protein, 1 to 1.5 per cent oil, 3.5 to 4.5 per cent fibre, 4.5 to 5.5 per cent ash and 62 to 65 per cent carbohydrate (Meena et al., 2021). The crop also suffers from many diseases caused by fungi, bacteria, viruses, nematodes and abiotic stresses (Chaudhari and Gohel, 2016). Anthracnose (Colletotrichum lindemuthianum Sacc. and Magnus), the most economically significant of all diseases, reduced green gram yield notably (Khaire and Hake, 2018) which causes severe leaf spotting known as “shot holes” progressing to defoliation and significantly reduces yield. Infected pods harm the seeds and affect germination. Due to anthracnose, the average seed yield losses and average stalk yield losses are 40.18% and 46.90% respectively (Sunil et al., 2009). Anthracnose in green gram is more common during the kharif season in south Gujarat due to predominant favourable weather conditions throughout the crop season. Contemplate to the emerging and devastating nature of the disease and economic loss of the crop in Gujarat, the current study was planned to assess fungicides for the management of anthracnose in south Gujarat.

Fungal pathogen isolation and purification
 
The diseased sample was collected from Pulse Research Station, NAU, Navsari and a microscopic examination was carried out in the lab. The diseased sample was divided into small explants, along with some healthy tissues and surface sterilised with a solution of 0.1 per cent mercuric chloride for one minute followed by three washes with sterile distilled water. Then the explants were placed on potato dextrose agar (PDA) plates in an aseptic condition and incubated at 28±2°C. The fungal growth was transferred aseptically to PDA slants and purified using the hyphal tip method after seven days of incubation.
 
In Vitro evaluation of various fungicides against C. lindemuthianum
 
The Poison Food Technique was employed to evaluate various fungicides in vitro against C. lindemuthianum. In vitro evaluation of different systemic fungicides, contact fungicides and combi-fungicides against C. lindemuthianum was carried out at the laboratory, Department of Plant Pathology, N.M. College of Agriculture, Navsari Agricultural University, Navsari, Gujarat. Poisoned media was prepared by blending different concentrations of respective fungicides with 100 ml of PDA. Seven days old test pathogen culture disc (5mm) was placed in the centre of the poisoned media plate whereas the plate without fungicides was kept as control. The inoculated plates were then incubated at 28±2°C. After 7 days of incubation, mycelial growth (mm) and growth inhibition (%) were observed and recorded. The per cent growth inhibition (PGI) of the pathogen in each treatment was calculated by the formula given by Vincent (1947):
   
Where,
PGI = Per cent growth inhibition.       
C = Colony diameter (mm) in control plate.
T = Colony diameter (mm) in treated plate.
 
Testing effective chemicals in pot condition against anthracnose of green gram
 
A glasshouse experiment was conducted to test the effectiveness of fungicides during their respective tests. The fungicides found effective in in vitro tests were used in pots individually for the management of anthracnose of green gram under greenhouse conditions. The healthy seeds of green gram were surface sterilized with 0.1 per cent mercuric chloride (HgCl₂) solution and washed thoroughly with sterile distilled water to remove traces of the mercuric chloride. Surface sterilized seeds were dipped in the spore suspension (1.0 × 10⁶ spores/ml) for 30 minutes and were sown in pots filled with autoclaved soil. The design used was a Completely Randomized Design (CRD). The number of replications was three and the number of treatments was seven (Two systemic, two non-systemic and two combi-fungicides were tested along with control). Mancozeb @ 3.0 g/kg, carbendazim 12% + mancozeb 63% @ 3.0 g/kg, were used as seed treatment before sowing of green gram seeds. Carbendazim @ 1.0 g/l, propiconazole @ 2.0 ml/l, captan 70% + hexaconazole 5% @ 0.6 g/l and chlorothalonil @ 3 g/l were used as foliar sprays twice at 30 days and 45 days after germination whereas control with only distilled water. Observations were recorded using the following formula given by Wheeler (1969):      
 

Effect of different systemic fungicides on growth inhibition of C. lindemuthianum 
  
To assess the systemic fungicide effectiveness against C. lindemuthianum in vitro, various concentrations (100, 250, 500 ppm) of carbendazim (50 WP), carboxin (75 WP), propiconazole (25 EC), azoxystrobin (23 EC), hexaconazole (5 EC) and  thiophanate methyl (70 WP) were tested. After fifteen days of incubation, observations were taken on mycelial growth and the percentage of growth inhibition [Table 1, Fig 1, 4(a)]. The majority of the above mentioned fungicides were found to be effective in preventing mycelial growth of C. lindemuthianum over control. But within each concentration, a significant difference was observed. Carbendazim showed the highest growth inhibition percentage (96.67%; 97.30%; 100%) over control at all different concentrations used (100, 250 and 500 ppm) respectively. Surprisingly, propiconazole showed absolute inhibition at 500 ppm. The poor inhibition was depicted by  hexaconazole and azoxystrobin at all different concentrations (Fig 1). It was discovered that the growth inhibition percentage and increase in the chemical concentration were positively correlated. At 500 ppm, carbendazim and propiconazole did not allow mycelial growth of the pathogen and significantly reduced the growth at 100 and 250 ppm. As a result, both fungicides were   more efficient against C. lindemuthianum. The remaining fungicides, however, were found to be comparatively moderate and less effective. In comparison to other fungicides, hexaconazole was found to be the least effective at all concentrations.
 

 

 
The above results are following Hedge (1998), who found that carbendazim effectively prevented chilli fruit rot brought on by C. capsici. In vitro testing of fungicides against C. dematium was done by Varaprasad (2000) and systemic fungicides carbendazim (0.1%) and kitazin (0.3%) were found to be effective at preventing fungal growth. Ten fungicides were tested in vitro by Madhusudhan (2002) against C. truncatum. Both benomyl and carbendazim, at concentrations of 0.025, 0.05 and 0.1%, inhibited fungal mycelium. Propiconazole, hexaconazole and carbendazim were also reported to be effective fungicides against green gram anthracnose by Joshi and Tripathi (2002) and Laxman (2006).
 
Effect of different non-systemic fungicides on growth inhibition of C. lindemuthianum
 
To assess the non-systemic fungicide effectiveness against C. lindemuthianum in vitro, various concentrations (1000, 2000, 2500 ppm) of copper oxychloride (50 WP), mancozeb (75 WP), chlorothalonil (75 WP), zineb (75 WP) and sulphur (80 WP) were tested. After fifteen days of incubation, observations were made on the average colony diameter and the per cent growth inhibition (Table 1, Fig 2, 4(b)). Mancozeb showed significantly higher per cent of growth inhibition (54.25%; 64.44%; 84.44%) at all concentrations (1000; 2000; 2500 ppm) respectively.  Significantly higher percentage of growth inhibition were observed with zineb (54.72%) and chlorothalonil (53.33%) at 1000 ppm concentration. Comparatively  sulphur (27.22%; 38.89%; 50.00%) showed less effectiveness against C. lindemuthianum at all concentrations (1000; 2000; 2500 ppm) respectively. The percentage of growth inhibition and the increase in chemical concentration were noticed to be positively correlated.
 

 

       
Comparable discoveries were made by Hedge (1998), who found that the non-systemic fungicide mancozeb at a concentration of 3000 ppm effectively controlled the C. capsici that causes chilli fruit rot. In vitro testing of fungicides against chickpea anthracnose (C. dematium) was done by Varaprasad (2000) and mancozeb (@ 0.2 and 0.3%) in the contact group of fungicides was found to be effective in inhibiting fungal growth. Mancozeb was also found to be the most effective contact fungicide against C. trancatum (Laxman, 2006).
 
Effect of different combi-fungicides on growth inhibition of C. lindemuthianum
 
Six combi-fungicides, which include carbendazim (12%) + mancozeb (63%), carboxin (37.5%) + thiram (37.5%), captan (70%) + hexaconazole (5%), hexaconazole (4%) + zineb (68%) and pyraclostrobin (5%) + metiram (55%), were in vitro evaluated for their effectiveness against C. lindemuthianum. The observation of average colony diameter and the percentage of growth inhibition was noted (Table 1, Fig 3, 4(c)). Carbendazim 12% + mancozeb 63% showed higher growth inhibition (85.64%; 88.80%) over control at 100 and 250 ppm respectively. The fungicides captan (70%) + hexaconazole (5%) (@ 55.56%) and carboxin (37.5%) + thiram (37.5%) (@ 55.36%) were found to be the least effective in preventing the growth of C. lindemuthianum at 100 ppm and captan (70%) + hexaconazole (5%) (75.27%) and hexaconazole 4% + zineb 68% (74.35%) at 250 ppm. Surprisingly, Captan 70% + hexaconazole 5EC (93.52%) emerged as effective in controlling pathogens than other fungicides at 500 ppm concentration.  At 100 and 250 ppm, pyraclostrobin 5% + metiram 55% significantly outperformed the control of the pathogen, but both pyraclostrobin 5% + metiram 55% (83.24%) and hexaconazole 4% + zineb 68% (83.33%) were significantly less effective against C. lindemuthianum at 500 ppm. Growth inhibition percentage and chemical concentration increase was revealed to be positively correlated.
       

 
Above findings are in accordance with Madhusudhan (2002), who found that SAAF (carbendazim 12% + mancozeb 63%) significantly reduced the growth of C. trancatum at 0.25% and 0.5%, respectively, by 99.22 and 85.92%. Chaudhari and Gohel (2016) reported carbendazim (12%) + mancozeb (63%) (1000, 2000, 2500 ppm) completely inhibited the mycelial growth and thus appeared significantly superior against C. gloeosporioides causal  fungus of green gram anthracnose.
 
Testing of effective fungicides in glasshouse
 
The potency of the effective fungicides discovered in vitro assay was further assessed in glasshouse experiment. Two systemic fungicides, carbendazim and propiconazole, two non-systemic fungicides, mancozeb and chlorothalonil, two combi-fungicides, captan 70% + hexaconazole 5% and carbendazim 12% + mancozeb 63%, were tested with a control in a pot experiment under glasshouse conditions. For the management of green gram anthracnose, seed treatment and foliar spraying were used and it showed that all treatments significantly reduced the severity of the anthracnose of green gram disease. The observation after 7 days of treatment on per cent disease index was noted (Table 2, Fig 4d). The lowest percentage of disease index was found in two foliar sprays of carbendazim 50 WP @ 1.0 g/l (10.70%), propiconazole 25 EC @ 2.0 ml/l (13.34%) and captan 70% + hexaconazole 5% @ 0.6 g/l (26.22%) spaced 15 days apart. On the other hand, seeds treated with carbendazim 12% + mancozeb 63% @ 3.0 g/kg seed (24.03%) and mancozeb 75 WP @ 3.0 g/kg seed (35.55%) had the lowest disease index compared to the control (69.86%).
 

       
Many researchers conducted similar experiments in order to manage green gram anthracnose. Bharadwaj and Thakur (1991) used carbendazim (0.1%), captafol (0.25%) and mancozeb (0.25%) for managing leaf spot and pod blight of urd bean caused by C. dematium under field condition. Varaprasad (2000) employed an integrated approach for the control of chickpea blight disease. Seed treatment with carbendazim @ 2 g/kg + two foliar sprays of SAAF (@ 0.05%) at 15 days intervals displayed the maximum reduction in disease incidence. Joshi and Tripathi (2002) reported that seed treatment followed by two prophylactic sprays of bavistin or tilt @ 0.1% each at 15 days intervals showed minimum disease severity and maximum grain yield followed by contaf (0.1%) and indofil M-45 (0.2%) to manage black gram anthracnose. Chauhan et al., (2014) conducted an efficacy test of fungicides against chilli anthracnose caused by C. capsici. The minimum disease intensity of 19.67% was reported in carbendazim at 0.05% with two foliar sprays, sprayed at 15 days interval which was statistically at par with spraying of 0.2% mancozeb (20.71%), 0.20% carbendazim + mancozeb (22.5%) and 0.20% copper oxychloride (23.16%).

Fungicide testing provides preliminary information on the efficacy of fungicides against pathogens in a short period, serving as a guide for field testing. In vitro studies found that carbendazim, propiconazole, mancozeb, chlorothalonil, captan 70% + hexaconazole 5%, and carbendazim 12% + mancozeb 63% were effective fungicides. Two systemic fungicides stood first in controlling pathogens at 500 ppm, followed by combi-fungicides and non-systemic fungicides. Foliar sprays of carbendazim 50 WP @ 1.0 g/l, propiconazole 25 EC @ 2.0 ml/l, and captan 70% + hexaconazole 5% @ 0.6 g/l spaced 15 days apart in a glasshouse had the lowest disease index. Foliar sprays can be categorized as “systemic” or “non-systemic”. Systemic fungicides, like Carbendazim, interfere with DNA biosynthesis during fungal cell division, but their persistence and resistance development can lead to resistance. Combi-fungicides are a good option for managing anthracnose of green gram in south Gujarat.

None.