Management of Wilt Complex of Chickpea with Seed Biopriming and Soil Application of Trichoderma spp.

Chickpea (Cicer arietinum L.) is the largest produced food legume of South Asia and the third-largest produced food legume globally (Suhasini et al., 2009). Nearly 172 pathogens (67 fungi, 3 bacteria, 22 viruses and 80 nematodes) have been reported infecting chickpea world-wide (Nene et al., 1996), but only a few of them have the potential to devastate the crop. Among them, the chickpea wilt complex is considered the most vital, devastating and challenging disease. The problem is widespread in several countries of the world like India, Iran, Pakistan, Nepal, Burma, Spain, Mexico, Peru, Syria and the USA (Nene et al., 1989; Jalali and Chand, 1992).
       
Wilt complex is caused by several soil-borne pathogens, among them, Fusarium wilt [Fusarium oxysporum Schlechtend.: Fr. f. sp. ciceri (Padwick) T. Matuo and K. Satõ], black root rot [Fusarium solani (Mart.) Sacc.], dry root rot [Macrophomina phaseolina (Tassi) Goidanich], wet root rot [Rhizoctonia solani Kühn] and collar rot [Sclerotium rolfsii Sacc.] are of considerable importance (Nene et al., 1981). Management of wilt complex of chickpea is difficult to achieve as the pathogens are soil-borne, surviving through resistant structure i.e. chlamydospores and sclerotia in the soil for years even in the absence of host and the crop remains susceptible throughout all the growth stages (Haware et al., 1986; Kaiser et al., 1994).
       
The use of chemical fungicides for the effective management of these pathogens is not possible because of the physical heterogeneity of the soil, which might prevent effective concentrations of the chemical from reaching the target pathogen (Tewari and Mukhopadhyay, 2001). To overcome such issues, biological control is one of the best, low-cost and ecologically sustainable methods. Also, the adverse effects of chemicals used in agriculture over decades have changed the mindset of farmers and consumers who are now producing and buying organic foods for their health (Vyas et al., 2019). Among various biocontrol agents (BCAs) examined against the plant pathogenic fungi, Trichoderma spp. has been widely studied for its biocontrol ability (Sharma et al., 2014).
       
A seed is the preferred technique of agriculture scientists for disseminating any technology as it is an easy means of adaptation. Seed biopriming is a comprehensive approach to agricultural sustainability. It allows rapid seed colonization by beneficial organisms and more uniform coverage of seed surface compared to the other techniques and also very effective technology in suppressing many diseases caused by seed and soil-borne pathogens (Sabalpara, 2015).
               
Because of the complex nature of the disease and based on the above facts, the present experiment was conducted to examine the performance of three important species of Trichoderma (T. viride, T. harzianum and T. asperellum) through seed biopriming alone as well as soil application of bioagents with enrichment of FYM for the disease management and yield parameters of chickpea in pot and field conditions during Rabi 2018-19 and 2019-20.

Isolation of the pathogens and collection of Trichoderma spp.
 
Samples of chickpea root and stem showing characteristic symptoms were collected from infected chickpea fields of Gujarat. A portion of each diseased root/stem/bark tissue of the sample was surface-sterilized with 1% sodium hypochlorite for 30 sec. and incubated on Potato Dextrose Agar (PDA). Among the different isolates three fungi viz., Fusarium oxysporum f. sp. ciceri, F. solani and Macrophomina phaseolina were isolated.
       
Different Trichoderma spp. for the experiment were obtained from the Department of Plant Pathology, BACA, Anand Agricultural University, Anand, the Department of Plant Pathology, CoA, Junagadh Agricultural University, Junagadh and the Department of Plant Pathology, NMCA, Navsari Agricultural University, Navsari.
 
Pot trial (In vitro)
 
The experiment was conducted at the Department of Plant Pathology, B. A. College of Agriculture, AAU, Anand during rabi 2019-20 in Completely Randomized Design with nine treatments along with three replications using variety Gujarat gram 2. Sterile soil was filled in 30 cm earthen pots. The chickpea seeds were treated with a suspension of talc-based formulation of respective Trichoderma spp. (2 x 108 CFU/g) @ 50 g product/ 250 ml of water/kg of seed. The bioprimed seeds were shade dried. Ten bioprimed seeds of chickpea were sown in each pot. For pathogen treated check sick pot technique developed by Nene et al., (1981) was followed. In which the fungi were grown on a 9:1 sand maize meal medium and added at 100 g/2 kg of soil.
       
The observations on seed germination (%), growth parameters i.e. shoot length (cm) and weight (g), root length (cm) and weight (g), vigour index at 15 DAS and seedling mortality (%) were recorded.

   
Vigour index (Abdul Baki and Anderson, 1973) was calculated as follows.
 
Vigour index = (Mean root length + Mean shoot length) x Germination (%)
 
Seedling mortality was calculated (Pande et al., 2012) as follows.  
 Field trials (In vivo)
 
The field trials were conducted at Agronomy Farm, BACA, AAU, Anand (22°35’N, 72°-55’E) during rabi 2018-19 and 2019-20 in randomized block design with eight treatments along with three replications using variety Gujarat gram 2. The crop was sown with 30 x 10 cm spacing having a gross plot size of 5.0 x 3.0 m and a net plot size of 4.2 x 2.4 m. The seed rate was used at 60 kg/ha. Chickpea seeds were bioprimed similarly as mentioned in pot trial and soil application of respective Trichoderma spp. enriched well-decomposed FYM (10 g/kg FYM) @ 100 g/m² of soil was done as per the treatments. A pathogen-treated check and an untreated control were also maintained. The observations on seed germination (%), disease incidence (%), seed yield (kg/ha), disease control (%) and yield increase over control (%) were recorded.
   
The per cent disease control was calculated by the following formula given by Wheeler (1969).
   
The per cent yield increase over control was calculated as,
 
   
       
The data on all the parameters were analyzed by DNMRT in both of the trials and the economics of the treatments was calculated.

Pot trial (In vitro)
 
The data presented in Table 1 revealed that maximum root length (13.90 cm), root weight (0.97 g), shoot length (24.90 cm), shoot weight (1.95 g), vigour index (3362) and minimum seedling mortality (15.23%) was recorded in treatment T7 i.e. seed biopriming for 10 hrs. with the suspension of talc-based formulation (2x10⁸ CFU/g) of T. asperellum (AAU isolate) @ 50 g in 250 ml of water/kg of seed followed by T6 i.e. seed biopriming for 10 hrs. with the suspension of talc-based formulation (2x10⁸ CFU/g) of T. viride (NAU isolate) @ 50 g in 250 ml of water/kg of seed which also recorded better root length (12.50 cm), root weight (0.81 g), shoot length (22.00 cm), shoot weight (1.86 g), vigour index (2875) and seedling mortality (19.88%). While data revealed a non-significant result in the germination per cent was in the range of 83.33 to 86.67%.
 

       
It can be therefore concluded that besides disease control, biopriming has a positive impact on germination and producing vigorous plant shoot and root system, which is key for better production.
 
Field trials (In vivo)
 
The pooled data of the year 2018-19 and 2019-20 presented in Table 2 and Fig 1 revealed that treatment T4 i.e. seed biopriming for 10 hrs. with the suspension of talc-based formulation (2 x 10⁸ CFU/g) of T. viride @ 50 g in 250 ml of water/kg of seed followed by soil application of T. viride enriched FYM (10g/kg FYM) @ 100 g/m² of soil recorded highest germination (93.84%), lowest disease incidence (14.72%), highest disease control (69.50%) with the highest yield (1775 kg/ha) and maximum yield increase over control of (50.70%) followed by treatment T6 i.e. seed biopriming for 10 hrs. with the suspension of talc-based formulation (2 x 10⁸ CFU/g) of T. asperellum @ 50 g in 250 ml of water/kg of seed followed by soil application of T. asperellum enriched FYM (10g/kg FYM) @ 100 g/m² of soil which recorded better germination (92.03%), lower disease incidence (16.99%), disease control (64.79%) and yield (1727 kg/ha) as compared to the treated and untreated check.
 

 

       
Economics of various treatments (Table 3) indicated that the maximum net realization was obtained in treatment T4 (45000 ₹ /ha) followed by T6 (42600 ₹ /ha). Looking to the ICBR, the highest return was obtained in treatment T1 (1:24.57) followed by T3 (1:22.73), however, the efficacy of these treatments was moderate against wilt complex.
 

       
It is evident from the data that seed biopriming followed by soil application of Trichoderma spp. found significant for control of the disease as well as gaining higher yield as compared to their single-use.
       
Similar results were also found by earlier workers. Prasad et al., (2002) and Mehta and Gangopadhyay (2017) found seed treatment + soil application of bioagents provided better control as compared to their use as either seed or soil treatment alone in reducing wilt (F. oxysporum f. sp. ciceri), wet root rot (R. solani) of chickpea and dry root rot (M. phaseolina) of mungbean under artificial soil inoculation condition. Shamarao et al., (2002) also recommended T. viride as an eco-friendly approach for the management of chickpea wilt as seed treatment with T. viride (Co) + soil application recorded 12.1 per cent wilt incidence compared to control (42.7%).
       
Animisha et al., (2012), Manjunatha et al., (2013), Sayyad et al., (2015), Lakhran and Ahir (2018) and Jat and Kumawat (2020) also found T. viride more effective compared to other different bioagents. In the present experiment, T. asperellum was found at par with T. viride in many parameters which were supported by Nagamani et al., (2018) as they found seed treatment and soil application of T. asperellum recorded the lowest dry root rot disease incidence (12.93%) and highest yield (1751 kg/ha) of chickpea.

Chickpea wilt complex was effectively and economically managed under in vivo and in vitro conditions through seed biopriming of Trichoderma spp. Under in vitro conditions, T. asperellum (AAU isolate) recorded maximum root length and weight, shoot length and weight, vigour index and minimum seedling mortality. Whereas, under in vivo conditions, the pooled results of two years revealed that seed biopriming of T. viride or T. asperellum followed by soil application of T. viride or T. asperellum enriched FYM found significant for the disease management as well as higher yield. Here, the combined application of seed biopriming followed by soil application was found significant as compared to only seed biopriming.