Macrophomina phaseolina is a soil- and seed-borne fungal pathogen that affects pigeonpea, causing stem canker and significant yield losses. We evaluated different inoculation methods to prove the pathogenicity of M. phaseolina in pigeonpea under pot culture conditions using variety GT-104 at N. M. College of Agriculture, NAU, Navsari (Gujarat). The pathogen was mass-multiplied on sterile sorghum grains and inoculated using pin prick, tooth brush, and carborundum powder. Among the methods tested, the pin prick method showed the highest disease incidence (80%) with visible spindle-shaped lesions after 47 days of inoculation. The tooth brush method resulted in 50 per cent disease incidence after 52 days, while the carborundum powder method had the lowest incidence (33.33%) after 58 days of inoculation. The results confirmed that the pin prick and tooth brush methods were the most effective for establishing pathogenicity. This finding, proved these methods to be reliable for testing stem canker caused by M. phaseolina in pigeonpea.

Macrophomina phaseolina, pigeonpea, pathogenicity, stem canker.

Pigeonpea (Cajanus cajan L. Millspaugh) is an important legume crop of rainfed agriculture in the semi-arid tropics in India. It is affected by several abiotic (temperature, drought, water-logging, photoperiodism, mineral deficiency) and biotic stresses like (fungi, bacteria, virus, insects and storage pests) that limit the realization of true potential yield of pigeonpea. The first record of pathogen of dry root rot seems to be from India as M. cajani (Syndow and Butler 1916). It is synonym of M. phaseoli. The disease is also reported from Australia, Myanmar, Sri Lanka, Vietnam and Zambia (Nene and Sheila 1990). Macrophomina phaseolina is primarily a soil and seed-borne fungal pathogen that incites the disease by producing microsclerotia/pycnidia (Farr et al., 1995; Pun et al., 1998). It has a wide host range of more than 500 species from 75 families with heterogeneous host specificity i.e., the ability to infect monocots as well as dicot and can exhibit non-uniform distribution in the soil (Mayek-Perez et al., 2001; Su et al., 2001). The disease is often referred to as charcoal rot, due to the charcoal type discoloration imparted to the colonized plant tissues. Macrophomina infection causes both pre- and post-emergence plant mortality. Characteristic post-emergence symptoms include the development of spindle shaped lesions with dark border and light grey center covered with small pin head sized pycnidia. Sclerotia allow the fungus to survive for a prolonged periods of time in the soil (Baird et al., 2003). 

Under conditions of high humidity and warm weather the symptoms of the disease appear on the stem which starts from the base and proceeds upward towards the branches. The symptoms of the disease ranged from restricted spindle shaped lesions on the stems with light grey centres and brown margins covered with small pin head sized pycnidia. The lesions may crack longitudinally after end of the rainy season. Extended lesions causing drying of the entire plant. Dropping of the pod-loaded secondary branches is common in the upper part of the affected plants.

Mass Multiplication of M. phaseolina. For inoculation, M. phaseolina was mass multiplied on sterilized sorghum grains for varietal screening trial in pot culture studies. The sorghum grains were soaked overnight in ordinary water. The excess water was drained out. About 150 grams of soaked sorghum grains and 30 ml of water were taken in each 250 ml conical flask, plugged with cotton and sterilized in autoclave at 15 lbs psi at 121.6°C for 30 minute conical flask containing the sterilized media (sorghum grain) were inoculated with mycelial disc of M. phaseolina (5 mm diameter) and incubated at 28±1 ºC for seven days. The flasks were shaken at alternate days for uniform colonization of the grains. The produced inoculum was mixed with sterilized soil in pots before sowing in varietal screening trial in pot condition.

Pathogenicity Test of Stem Canker of Pigeonpea. This study was conducted at Plant pathology department, N.M. College of agriculture, Navsari Agricultural University. To know the pathogenic nature of Macrophomina phaseolina, pure culture obtained from pigeonpea stem sample were tested on healthy pigeonpea plants of cv. GT-104 raised in plastic pots. Total 6 seeds of cv. GT-104 were sown in each pot (60 cm diameter) containing FYM and sterilized heavy black soil. The mycelial suspension (1×103 CFU/ml) was prepared from the 10 days old culture in distilled sterilized water with homogenizer. The pots were watered regularly up to saturation in the morning and the inoculation was carried out in the evening hours. The inoculation was carried out by three different methods. 

∙ Pin prick method: Sterile-pins were dipped in fungal suspension and pricked immediately on stem at 3 to 4 places. 

∙ Tooth-brush-injury method: Sterile tooth brush dipped in the suspension and inoculated with gentle rubbing on stem at 2 to 3 places. 

∙ Injury by carborandum-powder: Stem was injured with carborandum powder (600 mesh) and uniform quantity of inoculums was placed at the injured area. 

In all above methods, the pathogen was inoculated on stem by rubbing sterile cotton swab dipped in mycelial suspension of M. phaseolina isolated from spotted pigeonpea stem. Suitable controls with only distilled sterile water spray were maintained. All the plants were then kept under moist condition, which wetted thrice a day to maintain sufficient humidity. Observations with regard to infection and symptoms development were recorded. The fungus was re-isolated from the inoculated diseased stem and the morphological and cultural characters were compared with those of M. phaseolina, which was previously isolated from diseased pigeonpea stem.

The results presented in Table 1 and Fig. 1 showed that the pathogenicity was proved positive in all the methods of inoculation. On standing plants, spindle shaped lesions were observed after 47 days of inoculation in pin prick inoculation method. In tooth brush method and carborundum powder method the first lesions symptoms were observed after 52 and 58 days of inoculation, respectively. Among all methods, maximum disease incidence (80%) was recorded in pin prick method followed by 50% in tooth brush method. The minimum disease incidence (33.33%) was recorded in carborundum powder method. The re-isolation from the artificially inoculated and infected pigeonpea plants yielded the culture of M. phaseolina identical and similar to original in all the respects. This proved that pin prick and tooth brush inoculation method were the quickest and most efficient methods for proving the pathogenicity of pigeonpea stem canker pathogen, M. phaseolina.

Table 1: Pathogenicity test of Macrophomina phaseolina on pigeonpea cv.GT-104 in pot condition by different methods.

Fig. 1. Different methods of pathogenicity test of Macrophomina phaseolina on pigeonpea plant.

Results of the present study on the pathogenicity of M. phaseolina conform with Chaudhari (2013); Jehani (2015) they also proved the pathogenicity of M. phaseolina on standing plant of pigeonpea, which were inoculated by pin prick method, carborundum powder and tooth brush method. Scientist revealed that pin prick and tooth brush inoculation method were the best methods for proving the pathogenicity. Also similar result found by Kumari et al. (2017); Desai et al. (2019) proved the pathogenicity using pin prick method.

The pathogenicity of M. phaseolina in pigeonpea in term percent disease incidence was proved that pin prick (80.00%) and tooth brush inoculation method (50%) were the quickest and most efficient methods for proving the pathogenicity of pigeonpea stem canker pathogen, M. phaseolina comparing to carborundum powder method (33.33%).

The future scope of this research on Macrophomina phaseolina in pigeonpea includes exploring advanced molecular techniques to understand pathogen-host interactions better and identify resistant pigeonpea varieties. Further investigations can focus on the genetic diversity of M. phaseolina strains to assess their virulence across different agro-climatic regions. Integrating these findings with precision agriculture techniques could help in developing targeted disease management strategies. Additionally, exploring bio-control agents and eco-friendly fungicides could provide sustainable alternatives for managing stem canker. This research can also expand to study the pathogen's impact on other legume crops, contributing to a broader understanding of its host range and facilitating the development of cross-crop disease resistance strategies.

Kapadiya R.K., Rakholiya K.B.  and Patel J.V.  (2024). Pathogenicity of Macrophomina phaseolina (Tassi) Goid. caused Stem Canker of Pigeonpea. Biological Forum – An International Journal, 16(9): 43-45.