Pigeonpea [Cajanus cajan (L.) Millsp.] is traditionally cultivated as an annual crop in semi-arid regions of the world. It is an important grain legume of Asia (especially, the Indian subcontinent), Latin America, Eastern and Southern Africa. Globally, it is grown on 5 million hectares (m ha) in about 82 countries of the world. Pigeonpea has a unique place in Indian farming and India accounts for about 90% of the global production. It is the second most important pulse crop next to chickpea, covering an area of around 5.05 m ha with production of 4.34 mt and productivity 859 kg/ha. Area in Maharashtra is about 1.34 M ha with production 1.37 m tones and productivity 1023 kg/ha. Maharashtra's contribution to percentage of India in area is 26.44% and in production is 31.49% (Anonymous, 2021-22).

Pigeonpea affected by several abiotic factor viz., water-logging, drought, temperature, photoperiodism, mineral deficiency and also biotic factor including fungus, bacteria, viruses, mycoplasma and nematodes may infect pigeonpea, which causes more than 100 diseases severe as biological limitation on productivity (Nene et al.,1989). The widest spread and destructive of which is Fusarium wilt (Fusarium udum Butler), Sterility mosaic and Phytophthora blight (Phytophthora drechsleri f. sp. cajani) which are important in India. Stem canker incited by Phoma cajani has emerging as one of the very severe diseases of pigeonpea as the effect of climate change in India.

The pathogen of the disease, Phoma cajani was first reported to occur on pigeonpea in Brazil (Rangel, 1915). In India, the disease occurs in the states of Maharashtra (Khune and Kapoor 1981), Gujarat (Kannaiyan et al., 1981) and Madhya Pradesh (Agrawal and Nema 1985). Phoma stem canker was first observed in the Vidarbha region on local varieties of pigeonpea at Pulse Research Station, Dr. PDKV, Akola (Khune and Kapoor 1981).

The P. cajani is sporadic but can occasionally become extremely severe in favourable climatic condition. When the crop is in the flowering stage from November to February, it become more severe in dry weather and at low temperatures. It was a minor disease but now become increasingly serious in recent years, inflicting greater economic losses. The infection was 18.5% in 1878-79, 22.4% in 1970-80 and 38.0% in 1980-81 (Somani et al., 1985) and in Madhya Pradesh incidence was ranged between 5 to 78% in different farmer's field (Agrawal and Nema 1985), also in Odisha, during the periodic surveys and critical inspections, the disease was frequently observed in farmer's fields and experimental plots, where it caused 5 to 50 percent of plant death in mature plants (Behera et al., 2017). Recently an intensive roving survey was conducted to assess the incidence of phoma stem canker in the major pigeonpea growing districts of Vidarbha region in which disease incidence was noticed in all the locations surveyed with the range from 18.00 to 60.33 percent (Dhanorkar et al., 2024).

The in vitro efficacy of commercial fungicides against pigeonpea stem canker caused by Phoma cajani is crucial due to the growing threat this disease poses to pigeonpea cultivation. Stem canker leads to significant yield losses, affecting both the livelihood of farmers and the agricultural economy. Currently, there is a notable lack of comprehensive information on effective management strategies for this disease, leaving farmers with limited options for control. Studying the efficacy of chemical treatments in vitro provides valuable insights into potential management approaches. Therefore, considering the acreage, yield losses and importance of the disease, formulated the trial on evaluation of fungicides against stem canker diseases to find out the efficient fungicides for the better management of stem canker disease.

The present experiment was conducted at Department of Plant Pathology, Post Graduate Institute, Dr. PDKV Akola (M.S.) during 2023-24. 

Isolation of the pathogen. Phoma isolates were collected from major pigeonpea growing area from different agro climatic zones of India. Isolations were made by cutting infected parts from the junctions of healthy and diseased leaf regions and surface sterilizing with 70% ethanol. Sterilized bits were transferring to a petri plate containing sterilized PDA (Potato Dextrose Agar) media. The cultures of Phoma isolates were maintained on PDA (Hi-Media, Mumbai) slants stored at 4ºC for further study. 

Purification of the Pathogen. The fungus was further purified by single hyphal tip method. They are grown by inoculating in the centre of a plain agar plate. The fungus spreads out with its hyphal strands in search of nutrients. These hyphal strands could be located under low power of the microscope and the isolated hyphal tips marked. These tips were carefully transferred to potato dextrose agar slants to obtain the pure cultures of Phoma cajani. The culture was maintained by sub-culturing on potato dextrose agar medium at room temperature. 

Pathogenicity of the pathogen. Artificial inoculation of the pathogen was conducted to prove the pathogenicity of P. cajani on pigeonpea by applying spray inoculation with spore and mycelial suspension of the pure culture of P. cajani on the seedling of susceptible cultivar of Maruti (ICP 8863) in earthen pots. Phoma cajani spore suspension of 104 µL spores was taken from the 10-day- old culture and inoculated on susceptible cultivar of Maruti (ICP 8863)   in earthen pots. A control was separately maintained in which all the operations were similar except the addition of the fungal culture. Symptom appearance was observed at regular intervals (Somani et al., 1985).

In vitro evaluation of fungicides. Antifungal efficacy of fourteen fungicides were carried out against mycelial growth of P. cajani by poisoned food technique (Nene and Thapliyal 1993) using Potato dextrose agar as basal medium. Based on the active ingredient, an appropriate quantity of the fungicides was added in previously sterilized 100 ml PDA separately in 250 ml conical flasks. The flasks were shaken well to ensure uniform distribution of fungicides in the basal medium. Twenty ml of the medium containing fungicides was poured into sterilized petri dishes. After solidification, the plates were inoculated by the fungal disc of 5 mm diameter cut out from seven days old culture and incubated at 27±2 ℃ for seven days. The colony diameter of culture was recorded when plates under control were fully covered. The efficacy of fungicides was expressed as per cent inhibition of mycelial growth over control, which was calculated by using the following formula (Vincent, 1947).

Isolation and pathogenicity of the pathogen. Pigeonpea plants showing typical symptoms of stem canker were used to isolate P. cajani using standard tissue isolation techniques and incubated on a PDA medium at 25 ± 2°C for isolation of the causal organisms. Pure cultures of the pathogens were obtained by hyphal tip technique and were used for identification and further investigations. Based on the cultural and morphological characteristics, the pathogen was identified as P. cajani by using the taxonomic monograph of phoma identification manual (Boerema et al., 2004) and other published literature.

Pathogenicity test was carried out on susceptible cultivar Maruti (ICP8863) by seedling inoculated with spore suspension method. After incubation period, lesions with grey centres and dark brown margins are the first observed on stems. Koch’s postulates were confirmed by reisolating the fungus from diseased stem and compared with the original test fungus.

In vitro Evaluation of Fungicides. Even though plant disease management starts with the basic principles of management practices like exclusion and eradication, the chemical method would be a rapid method for the management of disease in crops. Disease management by means of fungicide usage is the most predominant practice. It has become inevitable to go for the management of phoma stem canker through fungicides. The fungicides, their concentration and mode of action were considered as factors for inhibiting the growth of the fungus. To identify a suitable fungicide and its effective concentration, non-systemic, systemic and combi-fungicides were evaluated for their efficacy against P. cajani under laboratory conditions. 

Most fungicides exhibit both fungistatic and fungicidal activities. Fourteen fungicides were tested at different concentrations including four systemic fungicides (Carbendazim 50% WP, Hexaconazole 5% SC, Tebuconazole 25.9% EC, Myclobutanil 10% WP), three contact fungicides (Captan 50% WP, Propineb 70% WP and Copper Oxychloride 50% WP) and seven combi fungicides (Carboxin 37.5% + Thiram 37.5% DS, Carbendazim 12% + Mancozeb 63% WP, Metalaxyl-M 4%+ Mancozeb 64% WP, Azoxystrobin 18.2% + Difenoconazole 11.4% SC, Propiconazole 13.9% + Difenoconazole 13.9% EC, Tebuconazole 50% + Trifloxystrobin 25 % WG and Azoxystrobin 4.7% + Mancozeb 59.7% + Tebuconazole 5.6% WG) each at 0.05, 0.10, 0.15, 0.20 and 0.25% conc. were evaluated against the P. cajani. 

Radial mycelial growth. Results revealed that, all the fungicides tested against isolates exhibited a wide range from 0.00 to 65.33 mm radial mycelial growth of P. cajani over untreated control. 

All fungicides tested, exhibited a wide range of radial mycelial growth of P. cajani, which was found to be decreased drastically with increase in their concentration. Radial mycelial growth/colony diameter of P. cajani was completely restricted by fungicides Tebuconazole 25.9% EC and Azoxystrobin 4.7% + Mancozeb 59.7%+ Tebuconazole 5.6% WG (each @ 0.05, 0.10, 0.15, 0.20 and 0.25% conc.), Carboxin 37.5% + Thiram 37.5% WP(@ 0.10, 0.15, 0.20 and 0.25% conc.), Hexaconazole 5% SC, Metalaxyl-M 4%+ Mancozeb 64% WP, Azoxystrobin 18.2% + Difenoconazole 11.4% SC and Tebuconazole 50% + Trifloxystrobin 25 % WG (each @ 0.15, 0.20 and 0.25% conc.)  and Myclobutanil 10% WP and Carbendazim 12% + Mancozeb 63% WP (each @ 0.20 and 0.25% conc.) followed by Carbendazim 12% + Mancozeb 63% WP at 0.15% (6.00 mm), Carboxin 37.5% + Thiram 37.5% WP, Hexaconazole 5% SC at 0.05%, Tebuconazole 50% + Trifloxystrobin 25 % WG at 0.10% and Propineb 70% WP at 0.25% (6.67 mm), respectively against maximum mycelial growth (80.00 mm) in untreated control.

Mycelial growth inhibition. Among fungicides tested, radial per cent mycelial growth inhibition recorded with the fungicides ranged from 18.34 to 100%. The cent per cent mycelial growth inhibition over untreated control was found with fungicides Tebuconazole 25.9% EC and Azoxystrobin 4.7% + Mancozeb 59.7% + Tebuconazole 5.6% WG (each @ 0.05, 0.10, 0.15, 0.20 and 0.25% Conc.), Carboxin 37.5% + Thiram 37.5% WP(@ 0.10, 0.15, 0.20 and 0.25% Conc.), Hexaconazole 5% SC, Metalaxyl-M 4%+ Mancozeb 64% WP, Azoxystrobin 18.2% + Difenoconazole 11.4% SC and Tebuconazole 50% + Trifloxystrobin 25 % WG (each @ 0.15, 0.20 and 0.25% conc.)  and Myclobutanil 10% WP and Carbendazim 12% + Mancozeb 63% WP (each @ 0.20 and 0.25% conc.) followed by Carbendazim 12% + Mancozeb 63% WP at 0.15% (92.50%), Carboxin 37.5% + Thiram 37.5% WP, Hexaconazole 5% SC at 0.05%, Tebuconazole 50% + Trifloxystrobin 25 % WG at 0.10% and Propineb 70% WPat 0.25% (91.66%), respectively. The least mycelial growth inhibition (18.34%) with Copper Oxychloride 50% WP at 0.05% conc. 

Average radial per cent mycelial growth inhibition recorded with the fungicides tested ranged from 40.50 to 100.00%. However, it was significantly highest and cent per cent mycelial growth inhibition with fungicide Tebuconazole 25.9% EC and Azoxystrobin 4.7% + Mancozeb 59.7% + Tebuconazole 5.6% WG (each @ 0.05, 0.10, 0.15, 0.20 and 0.25% conc.), Carboxin 37.5% + Thiram 37.5% DS(@ 0.10, 0.15, 0.20 and 0.25% conc.), Hexaconazole 5% SC, Metalaxyl-M 4%+ Mancozeb 64% WP, Azoxystrobin 18.2% + Difenoconazole 11.4% SC and Tebuconazole 50% + Trifloxystrobin 25 % WG (each @ 0.15, 0.20 and 0.25% conc.) and Myclobutanil 10% WP and Carbendazim 12% + Mancozeb 63% WP (each @ 0.20 and 0.25% conc.), respectively. The mean least mycelial growth inhibition (40.50%) was with Copper oxychloride 50% WP. 

In vitro evaluation of fungicides provides useful preliminary information regarding its efficacy against a pathogen within the shortest period of time and therefore serves as a guide for further testing. No systemic fungicides directly have contact with the diseased part of the plant and they are mostly multi-site inhibitors and are not absorbed by the plant and only stick to plant surfaces. These fungicides provide a barrier and that prevents the fungus from entering and damaging the plant tissues. Systemic fungicides translocate to plant parts and they are not covered by the application and protect the plant from inside. They are effective in smaller amounts and these fungicides are less prone to rain wash or photodegradation. The combi-fungicides are the combination of two fungicides with low risk and having multi-site action on the pathogen which will contribute to the avoidance of resistance development. The different fungicides in the mixture must be active against the target fungi so that subgroups that are resistant to one mode of action are controlled by the fungicide partner with a different mode of action.

The effectiveness of the triazoles fungicides in a combi form may be attributed to their interference with the biosynthesis of fungal sterols and inhibits biosynthesis of ergosterol. In many fungi, ergosterol is essential to the structure of the cell wall and its absence causes irreparable damage to the cell wall leading to the death of the fungal cell. A similar study was reported for the effectiveness of triazoles, which inhibit the biosynthesis pathway in fungi. Pethybridge and Hay (2001) revealed that fungicides such as Tebuconazole, Difenoconazole and Cyproconazole produced the most significant reduction in colony diameter of Phoma ligulicola due to methylation inhibition, which acts by inhibiting the C-14demethylation of 24-methylene dihydrolanosterol, aprecursor of fungal sterol biosynthesis. Tebuconazole 25.9% EC (at 0.10% conc.) inhibit cent per cent mycelial growth of Phoma spp. (Ingale et al., 2013).

Fig. 1. In vitro efficacy of fungicides against P. cajani isolates.

Plate 1. In vitro efficacy of fungicides against Phoma cajani.

Saju et al. (2011) revealed that Carbendazim 50% WP was significantly effective against Phoma leaf spot of large cardamom at all concentrations (0.05% - 0.15%) tested followed by Carbendazim + Mancozeb 75% WP (0.1% -0.4%). The Carbendazim 12% + Mancozeb 63% WP, Metalaxyl-M 4%+ Mancozeb 64% and Carboxin 37.5% + Thiram 37.5% WP reported to cause maximum mycelial growth inhibition in many Phoma spp. (Behera et al., 2013; Patil et al., 2010; Thesiya et al., 2019; Muthulakshmi and Seethapathy 2019; Nanjundaswamy et al., 2020; Pal et al., 2020). 

The mean minimum inhibition was recorded in Azoxystrobin 23% SC (39.63%) followed by Kitazin 48% EC (75.43%). Difenoconazole 25 EC (at 0.05%) inhibits cent per cent mycelial growth of Phoma exigua (Wani et al., 2022) and A. Rabiei (Javed et al., 2023).

(i) Future research can focus on identifying and developing pigeonpea varieties with genetic resistance to P. cajani, using advanced molecular techniques and traditional breeding approaches.

(ii) Investigating the use of biocontrol agents, such as Trichoderma or beneficial microbes, to manage stem canker in a sustainable and eco-friendly manner can be a key area for future study.

(iii) Understanding the environmental factors, pathogen biology and disease cycle that influence the spread and severity of P. cajani could help in predicting outbreaks and developing effective management strategies.