Integrated Management of Stem and Root Rot of Cowpea Caused by Macrophomina phaseolina (Tassi.) Goid. using Fungicides, Bioagents and Organic Manures

Cowpea (Vigna unguiculata L.) is one of the most ancient human food sources and short duration multipurpose pulse crop grown extensively in tropical and subtropical countries. It belongs to the family Fabaceae. Cowpea forms an important component of farming system, it fits well in a variety of cropping systems and is grown as a cover crop, mixed crop, catch crop or green manure crop in different states of India (Alexandre et al., 2016). Cowpea is grown across the world on an estimated 23.4 mha with a production of 18.29 mt and productivity of 637 kg/ha. In India cowpea is grown in an area of 4.00 mha with a production of 2.70 mt and productivity of 567 kg/ha (FAO, 2020). The cowpea crop is affected by number of fungal, bacterial and nematode diseases. Among Stem and root rot incited by Macrophomina phaseolina (Tassi.) Goid. has been rated as most devastating disease of cowpea which can cause yield loss up to 5 to 39 per cent (Mohanapriya et al., 2017; Gireesha et al., 2023). On cowpea, disease symptoms are clearly visible from the time of emergence and can be evaluated at various stages of development of the plant. In grown up plants, M. phaseolina causes lesions on stems, spikes, pods and seeds. On stems, lesions are beige and appear at the ramification point of lateral secondary branches. Colonized tissues become gray and covered with abundant minute black punctuations. Initially these punctuations are immersed, becoming gradually more prominent (Bouhot, 1967; Adam, 1986). The most striking symptom is the sudden wilting and drying of the whole plant while most of the leaves remain green. The stem and branches are then covered with black bodies and give the charcoal or ashy appearance of dead plants (Chan and Sackston, 1973). Withering can be observed from seedling to maturity stage and is the result of necrosis of roots, stems and mechanical plugging of xylem vessels by microsclerotia, but also by toxin production and enzymatic action (Kuti et al., 1997; Jones and Wang, 1997).
       
The fungus invades host both inter and intracellularly, it produces numerous microsclerotia on host tissue, which measure about 110-130 µ in diameter. Often the conidial or pycnidial stage is produced on the host (Nitharwal, 2019). The fungus is mainly a soil dweller and spreads from plant to plant through irrigation water, implements and cultural operation. The sclerotia and pycnidiospore may also become air borne and cause further spread of the pathogen (Rangaswami and Mahadevan, 2008). With the view to manage stem and root rot disease of cowpea, studies on different aspects comprising of disease management components such as testing of fungicides and biocontrol agents under in vitro was carried out by Singh and Srivastava (1988). A review of literature revealed that very limited information is available on management of this disease. By considering the increasing incidence of stem and root rot of cowpea and the economic losses caused by the disease, the present investigation was carried out to formulate suitable management practices against stem and root rot.

Preparation of giant culture of Macrophomina phaseolina
 
The pathogen, M. phaseolina was isolated from the stem and root rot infected cowpea plants collected from experimental plots of Department of Plant Pathology at Main Agricultural Research Station (MARS), University of Agricultural Sciences (UAS), Dharwad, Karnataka by tissue segment method on potato dextrose agar (PDA) medium. Sorghum seeds were used as substrate for giant culture preparation. The substrate was prepared by mixing 200 g of crushed sorghum seeds and 50 ml distilled water in 500 ml conical flask and sterilized at 15 psi for one hour for two consecutive days. Flasks was subsequently inoculated with 4-5 discs of seven days old culture of M. phaseolina and incubated at 28±1°C for 20 days (Choudhary et al., 2011). During incubation, the culture was mixed thoroughly to get uniform growth.
 
In vitro evaluation of fungicides
 
The efficacy of different seed dressing fungicides viz.,Carbendazim 50% WP, Captan 50% WP, Tebuconazole 5.4% w/w FS, Mancozeb 50% + Carbendazim 25% WP, Captan 70% + Hexaconazole 5% WP, Carboxin 37.5% + Thiram 37.5% DS, Thiophanate Methyl 45% + Pyraclostrobin 5% FS, Penflufen 13.28% w/w + Trifloxystrobin 13.28% w/w FS and Mancozeb 75% WP were tested under in vitro condition by poisoned food technique (Nene and Thapliyal, 1973) at 0.1, 0.2 and 0.25 per cent concentrations. Similarly, control was maintained by placing a mycelia disc of the pathogen at the centre of Petri plate containing the medium without any fungicide. The per cent inhibition of mycelial growth was calculated using the formula given by Vincent (1947).
       
The diameter of fungal colony was measured in each of the treatment when the pathogen growth in control plate was full. The colony diameter inhibited in fungicide treated plates as compared to control was taken as a measure of fungi toxicity. Per cent inhibition over control was calculated as per the formula given by Vincent (1947).
            
Where,
I= Per cent inhibition.
C= Colony diameter in control (mm),
T= Colony diameter in treatment (mm).
 
In vitro evaluation of biocontrol agents
 
The efficacy of available fungal and bacterial biocontrol agents viz., Trichoderma harzianum, Pseudomonas flourescens and Bacillus subtilis collected from the Institute of Organic Farming (IOF), University of Agricultural Sciences, Dharwad and Trichoderma viride from Multiplex Nisarga Company were evaluated under in vitro conditions against mycelial growth of M. phaseolina by dual culture technique (Dennis and Webster, 1971). Similarly, control was maintained by placing mycelia disc of the test fungus at the centre of Petri plate containing the medium devoid of any biocontrol agents. Percentage inhibition of mycelial growth was calculated as per Vincent (1947) formula.
       
Observations on colony diameter were recorded when the control plates were fully covered by pathogen and per cent mycelial growth inhibition was calculated as per the formula given by Vincent (1947) as discussed earlier.
 
Integrated disease management
 
Field experiment on integrated management of stem and root rot of cowpea was conducted at MARS, University of Agricultural Sciences, Dharwad during rabi 2021-22 and 2022-23 by using in vitro effective fungicides and biocontrol agents along with organic manures. The experiment was laid out in randomized complete block design (RCBD) with nine treatment schedules including untreated control and three replications. Cowpea variety C-152 was sown at 45 cm × 20 cm spacing with plot size of 2.25 m × 4 m and all the recommended package of practices was followed to raise the crop, except for disease management. Before sowing all the seed furrows were uniformly applied with mass multiplied inoculum of M. phaseolina on sterilized sorghum grain medium and the details of the treatments are given in Table 3.
       
In the field experiment, observations on seedling emergence at 10 days after sowing (DAS) was recorded by counting the number of seeds germinated and total number of seeds sown and per cent seedling emergence was calculated by the following formula:
   
Disease incidence was recorded at 30, 60 and 75 DAS by counting the total number of plants and number of plants infected and per cent disease incidence at all the above stages of plant growth was calculated by the following formula:
   
The plots were harvested separately and grain yield was recorded and further converted to quintal per hectare (q/ha). The grains collected from each treatment separately and hundred seed weight was recorded by using digital electronic balance.
       
The cost of production was analyzed in order to find out the most economic treatment of different management practices. Cost and return analysis were done according to the procedure of Kushwah et al., (2017) and Bhupender et al., (2020). The benefit cost ratio (BCR) was calculated as follows:
   
Effect of soil temperature and soil moisture on disease development
 
Observations on soil temperature and soil moisture were noted down from flowering to harvesting stage i.e. from 40 DAS to 90 DAS at 10 days’ interval (40, 50, 60, 70, 80 and 90 DAS) in untreated check plots (T₉) in integrated disease management treatment plots. All the three replications were considered for taking observations and the mean was calculated. Then the data were analyzed statistically. Soil temperature was recorded by using soil thermometer and soil moisture was estimated by digital soil moisture meter (Lutron PMS 714). The correlation was made between the soil temperature, soil moisture and per cent disease incidence of untreated check.
 
Data analysis
 
Data was analysed as per the procedures given by Panse and Sukhatme (1978). Data in percentage was converted to angular transformed values and √(X + 1) values, before analysis (Walter, 1967). Analysis of variance and least significance difference (LSD) were determined at 5 and 1 per cent probability. Treatment means were compared using LSD to determine efficacy of different treatments.

In vitro assay of seed dressing fungicides against Macrophomina phaseolina
 
Significantly maximum mean inhibition of mycelial growth of M. phaseolina was observed in mancozeb 50% + carbendazim 25% WP (97.59%) which was on par with carboxin 37.5% + thiram 37.5% DS (97.48%) followed by carbendazim 50% WP (92.44%) and tebuconazole 5.4% w/w FS (90.32%). Lowest mycelial inhibition of 60.85 per cent was recorded in captan 50% WP. Among the different concentration tested, maximum mycelial inhibition of 90.26 per cent was noticed in 0.25 per cent concentration and was significantly superior over 0.2 (84.09%) and 0.1 (79.33%) per cent concentrations (Table 1).
 

       
The findings align with previous research conducted by Nitharwal (2019), Pathak et al., (2019) and Kumari et al., (2022). Maruti et al., (2017), who reported that carbendazim 12 % + mancozeb 63 % WP and carboxin 37.5% + thiram 37.5% WP demonstrated complete inhibition of R. bataticola at all concentrations tested. Similarly, Sangappa and Mallesh (2016) also observed that carbendazim 12% + mancozeb 63% exhibited complete inhibition of mycelial growth at various concentrations, specifically at 0.05, 0.10 and 0.2 per cent. These consistent findings underscore the effectiveness of these fungicidal combinations against the pathogen.
 
In vitro assay of bioagents against Macrophomina phaseolina
 
In vitro evaluation of bioagents by dual culture technique revealed that there is significant difference in per cent mycelial growth inhibition of M. phaseolina by different bioagents tested. Maximum mycelial growth inhibition of 75.53 per cent was noticed with Trichoderma harzianum followed by T. viride (67.72%) and Pseudomonas fluorescens (61.41%). The least mycelial growth inhibition of 56.00 per cent was recorded with Bacillus subtilis (Table 2).
 

       
The results are in accordance with Kumar and Kelaiya (2021), Gajera et al., (2012) who evaluated the in vitro potentialities of Trichoderma species against M. phaseolina. The maximum growth inhibition of test pathogen was observed by antagonist T. koningi (74.3%) followed by T. harzianum (61.4%). Microscopic study showed that these two antagonists were capable of overgrowing and degrading the mycelium of M. phaseolina, coiling around the hyphae with apressoria and hook-like structures. The specific activities of cell wall degrading enzymes such as chitinase, β-1, 3 glucanase, protease and cellulase were also recorded (Silva et al., 2004 and Mukherjee et al., 2003). Rathore et al., (2020) and Lakhran and Ahir (2022) also reported the effectiveness of T. Viride, Bacillus subtilis and Pseudomonas fluorescence on radial growth of M. phaseolina.
 
Integrated management of stem and root rot of cowpea
 
Among the nine integrated treatment modules evaluated during rabi 2021-22 and 2022-23 revealed that, seed treatment with carboxin 37.5% + thiram 37.5% WS @ 2g/kg seeds (T₁) recorded significantly highest seed germination (95.17%), least per cent disease incidence (14.46) with highest grain yield (13.35 q/ha) and 100 seed weight (11.47 g). The treatment T₁ was on par with the treatment T₂ involving seed treatment with mancozeb 50% + carbendazim 25% WS @ 2 g/kg seeds (94.83% seed germination) (16.18 PDI) (12.67 q/ha) (10.88 g). Lowest per cent seed germination (78.00), highest per cent disease incidence (55.58), least grain yield (4.37 q/ha) and 100 seed weight (5.86 g) was recorded in untreated control (T₉) (Table 3a and 3b). Assessing the cost-benefit ratio is a crucial element of managing plant diseases economically. The findings in the table indicate that the highest benefit-to-cost ratio of 2.78 was achieved through the application of carboxin 37.5% + thiram 37.5% DS at a rate of 2 g/kg of seeds (T₁ treatment). Following closely was the seed treatment with Mancozeb 50% + Carbendazim 25% WP at the same application rate (T₂), which is of 2.66. In contrast, the untreated control (T₉) had the lowest cost-benefit ratio of 0.76.
 

 

       
The outcomes align with the findings of Jambhulkar et al., (2015), Nagamani et al., (2011) and Kullalli (2019). A research conducted by Sunkad et al., (2018), where it was reported that seed treatment involving Mancozeb 50% + Carbendazim 25% WS @ 3.5 g/kg, followed by soil drenching with the same fungicide (3 g/L), achieved the most substantial reduction in dry root rot incidence in chickpea, with the highest seed yield and a test weight. In a field study on integrated management of dry root rot in cowpea caused by Rhizoctonia bataticola, dry seed dressing with carbendazim was found most effective followed by seed treatment and soil application of T. viride combined with P. fluorescens enriched with FYM (Koli, 2019). Under field conditions, maximum root rot (M. phaseolina) reduction (83.76%) with highest pod yield of chickpea (19.5 q/ha) and net return (Rs 39,826/ha) was recorded in treatment involving seed treatment with tebuconazole 50% + trifloxystrobin 25% WG @ 1.5 g/kg along with soil application of T. harzianum @ 10 kg/ha (Malagi et al., 2023). Seed treatment with P. flourescens (10 g/kg) and soil application of neem cake (2.5 kg/ha) recorded the least root rot (M. phaseolina) incidence in cowpea (Vengadeshkumar et al., 2019).
 
Effect of soil temperature and soil moisture on disease development
 
The incidence of stem and root rot of cowpea with respect to variation in soil temperature and moisture was noted down from 40 DAS to 90 DAS in untreated control plots. The results indicated that there was increased incidence of disease due to increased soil temperature coupled with optimum soil moisture. Soil temperature of 36°C coupled with soil moisture of 29.88 per cent was most favourable for M. phaseolina infection which resulted in maximum disease incidence of 46.65 per cent (Table 4). Correlation studies was made between soil temperature, soil moisture and disease incidence. Results revealed that significant positive correlation (0.939) was observed between high soil temperature and disease incidence whereas, negative correlation (-0.995) was noticed with high soil moisture and disease incidence (Table 5).
 

 

       
The results were in conformity with Bashir (2017), Arora and Pareek (2013). Sharma and Pandey (2013) reported that rate of infection increases with higher soil temperature and low soil moisture because hot and dry soil conditions resulted into pathogen grow faster and produce large amount of microsclerotia that causes more infection.

Among the seed dressing fungicides evaluated against M. phaseolina, mancozeb 50% + carbendazim 25% WP and carboxin 37.5% + thiram 37.5% DS were found most effective. Among the bioagents tested, T. harzianum was the most effective in inhibiting the mycelial growth of M. phaseolina followed by T. viride (67.72%) and P. fluorescens (61.41 %). Among the nine integrated treatment modules evaluated during rabi 2021-22 and 2022-23 revealed that, seed treatment with carboxin 37.5% + thiram 37.5% WS @ 2 g/kg seeds (T₁) recorded significantly least per cent disease incidence (14.46) with highest grain yield (13.35 q/ha) and 100 seed weight (11.47 g) with B:C ratio of 2.78.

The authors declare that they have no conflict of interest.