Managing chickpea wilt; Fusarium oxysporum through use of biorationals

Among 37 pathogens attacking chickpea, in India, chickpea wilt, caused by Fusarium oxysporum f. sp. ciceri, is one of the most devastating and serious diseases of chickpea damaging it at all the stages, resulting in complete drying and causing significant losses. This disease is becoming increasing important in Jammu region of Jammu and Kashmir State. Fusarium oxysporum f.sp. ciceri, a soil borne pathogen colonizes the xylem vessels and completely block them to cause wilting, is one of the serious diseases of chickpea, causing losses upto 10-100% depending on environmental conditions (Sumitha and Gaikwad, 1995). Farmers resort to the application of chemical fungicides viz., Carbendazim, to manage this disease.
       
With the increased safety and health concerns, researchers are looking for alternatives to chemical pesticides, which are as good as them. Though pyrethroids, neem products are well established as pesticides, other plant products needs to be explored. Plethora of literature is available on fungicidal activities of several plant materials extracted with different solvents. Thousands of phytochemicals which have inhibitory effects on all types of microorganisms in vitro should be subjected in vivo testing to evaluate the efficacy in controlling the incidence of disease in crops, plants, and humans. Efficient collaborations with pharmacologists and medical doctors, plant pathologists and microbiologists are crucial to see the complete development of an interesting lead compound into an exploitable product (Gurjar et al., 2012).
       
Plant pathogenic fungi have caused devastating losses worldwide. Chemical fungicides are usually the first choice of the farmers, probably because of its ease of applicability and easy availability. Looking to the adverse impact of these chemical fungicides on environment, ecology and human beings, researches on finding effective alternatives need to be strengthened.
       
Jammu province is rich in floral diversity as it covers three agro-ecological zones (sub-tropical, intermediate and temperate). All the three zones exhibit plants unique to its climatic conditions. Keeping all these facts in mind, research was conducted with the objective to assess the various extracts and fractions of few promising plants against chickpea wilt, Fusarium oxysporum f sp. ciceri. The present study is an attempt to reduce, the pesticidal load and its ill-effects in the environment and ecological system.

All the three agro-ecological zones (sub-tropical, intermediate and temperate) of the Jammu province were surveyed for potential pesticidal plants (Table 1). The plants/plant parts were collected from Bari Brahmana, (latitude- 32.64°N, longitude-74.91°E and elevation-328.85 meters) Samba (latitude-32.55°N, longitude-75.11°E and elevation - 348 meters), Kathua (latitude - 32.39°N, longitude-75.52°E and elevation-387 meters), Mansar (latitude-32.70°N, longitude – 75.15°E and elevation – 666 meters) and Patnitop (latitude – 33.07°N, longitude – 75.34°E and elevation – 2024 meters), keeping the following points in mind.

-   The plant is abundant in the area.
-  It has some medicinal properties based on available literature and information gained from local people.
 

       
A total of 64 plants / plant parts were collected from all the three zones; sub tropical-23 (Table 2), intermediate-17 (Table 3) and temperate-24 (Table 4).
       

 

 

 
The collected plant material was shade dried and kept in plastic boxes for further use.
 
Preparation of plant extracts
 
Weighed quantity of shade dried plant material was crushed and kept in a round bottom flask. The solvent methanol was added to it in a volume just enough to immerse the bits. Refluxing was done by fitting the flask with a water condenser and boiling the set using heating mantle for 6 h (Fig 1). The extract was then filtered out of the flask and was concentrated by distillation process. This refluxing and distillation procedure was repeated thrice for the complete extraction of plant material. The quantity of extract obtained was also recorded (Fig 2). The methanolic extracts of all the collected plants / plant parts were then prepared by following the standard procedure of refluxing and distillation (Kumar et al., 2001).    
 

 

                
Test fungus
 
Fusarium oxysporum f.sp. ciceri was isolated from infected chickpea plant using standard pathological techniques. The media used was Potato Dextrose Agar (PDA). Pure culture of the test fungus was maintained. The assessment of fungitoxicity was done by poisoned food technique (Grower and Moore, 1962). Seven days old culture of the test fungus was used for the preparation of innoculum disc of 5 mm in diameter (Fig 3).
 

 
Antifungal assay
 
A volume of 0.5 ml of each concentration was asceptically poured into the petriplate followed by the addition of 9.5 ml of melted PDA and was swirled gently to achieve through mixing of the contents. Two controls, one treated with Carbendazim and the other completely untreated, were kept. In the control set, no extract was used. After the solidification of the media, one innoculum disc of the test fungus was aseptically inoculated upside down at the centre of the petriplate and incubated at 25 ± 2°C. The average diameter of the fungal colonies was measured on the 7^th day of incubation and percentage of mycelial growth inhibition was calculated (Rao and Srivastava, 1994). All the extracts were initially assessed at 1000 ppm and almost all recorded complete inhibition. The concentration was then reduced to 500 ppm and again most of the extracts showed hundred per cent inhibition. Finally the extracts were assessed at 200 ppm concentration.

 

Where,
g_c = growth of mycelial colony in control set after incubation   period substracting the diameter of innoculum disc.
g_t = growth of mycelial colony in treatment set after  incubation  period substracting the diameter of innoculum disc.
       
Based on the growth inhibition effects of these extracts on the test fungus, F. oxysporum f. sp. ciceri, few plants/plant materials were selected for further evaluation.
 
Statistical analysis
 
The data were subjected to analysis of variance (ANOVA) in a Completely Randomized Design after appropriate transformations as suggested by Gomez and Gomez (1984) before statistical analysis. The difference of two means between treatments exceeding Critical Difference (CD) value is significant (Panse and Sukhatme, 1978).

Among the 23 plants/plant parts methanolic extracts, evaluated against F. oxysporum, Boerrhavia diffusa root extracts recorded lowest radial growth (6.0 mm), followed by Boerrhavia diffusa stem extract (8.0 mm) and Achyranthes aspera root extract (9.0 mm). Accordingly, Boerrhavia diffusa stem and root extracts again exhibited 94.5 per cent inhibition as evident from Table 5. This was followed by Achyranthes aspera root (91.11%) and stems (86.67%) extract. Murrya koengii fruits (75.56%) and leaves (70.00%) extract also exhibited more than 70% inhibition. All the treatments exhibited higher percent inhibition than that in control (water), while most treatments effect were comparable to that of control (Carbendazim). Few (Boerrhavia diffusa root, Boerrhavia diffusa stem and Achyranthes aspera root extract) performed even better than the carbendazim treatment, recording almost complete inhibition.
 

 
Woodfordia fruticosa root extract recorded lowest radial growth of 20 mm among the intermediate zone plants, followed by Diplocyclos palmatus fruit (21 mm) and Nicotiana rustica leaves + stem (22 mm) (Table 6). Likewise, Woodfordia fruticosa roots (77.78%), Diplocyclos palmatus fruits (76.67%), Nicotiana rustica leaves + stem (75.56%), recorded more than 75.00% inhibition. Though all the extracts recorded  percent inhibition higher than the control (water), Woodfordia fruticosa root extract (20.0 mm radial growth), Diplocyclos palmatus fruits (21.0 mm), Nicotiana rustica leaves + stem (22.0 mm) and Vitex negundo leaves (30.0 mm), exhibited percent inhibition higher than carbendazim treatment (34.0 mm).
 

       
Lowest radial growth was recorded in Arisaema flavum root / tuber extract (4.0 mm) and stem + leaves extract (8.0 mm), among the temperate zone plants as evident from Table 7. This was followed by Coccinia grandis fruit (14 mm) and Verbascum thapsus leaves + stem extract (22 mm). Accordingly, Arisaema flavum roots/tubers extract also exhibited 95.55% and its leaves recorded 91.00% inhibition. This was followed by Coccinia grandis (84.44%) and Verbascum thapsus leaves + stem extract (75.56%).
 

       
Corroborating our study, few workers have reported the fungicidal activity of botanicals against chickpea wilt. Seed treatment with garlic leaf extract (Singh et al., 1979) and neem oil (Singh et al., 1980) are reported to reduce the pathogen. The antifungal effect of aqueous extracts of four plant species viz; Azadirachta indica A. Juss., Datura metel L. Torr., Ocimum sanctum L. and Parthenium hysterophorus L. was observed in vitro study. It was found that all four plant extracts at 40% concentration were effective in controlling the mycelial growth of F. oxysporum f. sp. ciceri (Irum, 2007). Leaf extract of Azadirachta indica at 100% conc. completely inhibited germination of pathogen spores (Singh and Hari Chand, 2004). Azadirachta indica leaf extract gave maximum inhibition (55.19%) of radial growth of F. oxysporum f. sp. ciceri (Hossain et al., 2013).
       
The antifungal or antibacterial activity of Verbascum Thapsus, Woodfordia fruticosa, Arisaema flavum, Coccinia grandis, have been proved by few researchers, which is in accordance with our study. Verbascum thapsus (Schrophulariaceae) better known as Mullein is a medicinal plant used in the treatment of inflammatory diseases, asthma, spasmodic cough, diarrhea and other pulmonary problems. Verbascum thapsus leaves were treated with n-hexane, chloroform, methanol, cold and warm water to obtained the extracts. In accordance with our studies, antifungal activity of V. thapsus was observed in its methanol extract (1000 µg mL^-1) against Fusarium graminearum and Macrophomina phaseolina. The antifungal studies of Coccinia grandis instant juice powder revealed a significant activity against fungal strains. It showed 2 to 5.1 mm zone of inhibition in the aqueous and solvent extract, due to the presence of phytochemicals (Elicy and Thilagavathi, 2017). Antibacterial activity of the flowers of Woodfordia fruticosa was assessed on different microorganisms (Kumar et al., 2015).
       
Arisaema flavum crude extract was active against all bacterial strains except Staphylococcus aureus. Maximum zone of inhibition (13.9 mm) was observed against Pseudomonas picketti. An average zone of 10-11 mm was observed against Micrococcus leutus, Bacillus subtilis, and Salmonella Setubal. Chloroform and methanol fractions of Arisaema flavum showed moderate activity against three strains while ethyl acetate fraction showed mild inhibition (9.6 mm) of Micrococcus luteus (Singh et al., 2004). Similarly, the crude extract of A. flavum was found active against different bacterial strains (three Gram positive and two Gram negative) (Bibi et al., 2011).

These potentially active plant extracts (Boerrhavia diffusa root and stem, Achyranthes aspera root, Woodfordia fruticosa root, Diplocyclos palmatus fruit, Nicotiana rustica leaves + stem, Arisaema flavum root / tuber and stem + leaves,  Coccinia grandis fruit, Vitex negundo leaves and Verbascum thapsus leaves + stem) may be further evaluated in vivo and utilized for managing plant diseases in fields. They can also be further developed into botanical pesticides, in similar lines as neem and exploited commercially. This shall help greatly in mitigating the climate change impact up to some extent.

The author is highly thankful to Department of Science and Technology, Govt. of India, New Delhi for providing financial support for the conduct of this research.