Novel Liquid Mycofungicide: An Ideal Approach in Protecting Tomato from the Damping-off Disease in Nursery

Background: A novel liquid formulation of Trichoderma asperellum was prepared and its effectiveness was assessed by different methods for the management of tomato damping-off in the nursery. The experiments were conducted for two consecutive years in a randomized block design with three replications. Results: T. asperellum formulation improved seed germination and management of tomato damping-off when compared with control. All the treatments performed better over control, in the rst year, the antagonist’s application enhanced seed germination up to 75.75 percent, however, it was 60.13 percent in control. There was 10.93 to 20.38 percent seedling mortality due to damping off which was comparatively lower than the control (26.98%). A similar trend of seed germination and disease incidence was observed in the second year. In addition to managing the disease, the antagonist certainly promoted the vegetative growth which was reected as increased shoot and root length in comparison to control during both years. During the rst year, shoot length ranged 10.90 – 12.85 cm as compared to the control (8.72 cm) and root length ranged from 3.21 to 3.65 cm which was greater than the control. Almost a similar trend in the vegetative growth parameters of seedling was observed during the second year. Conclusion: The present investigation showed that the tested antagonist’s formulation could eciently managed the tomato damping-off as well as encouraged the vegetative growth of seedlings which ultimately ensured better and healthy seedling. And this formulation can successefully used through different methods to take care of tomato damping off.


Background
Tomato (Solenum lycopersicum L.) is an imperative constituent of human food basket as a good source of vitamins and minerals. It is consumed as a fresh, vegetable as well as processed products such as juice, ketchup, sauce, canned fruits, puree, paste, etc. India occupied about 789 thousand ha under tomato with a production volume of approximately 19759 thousand MT. It is also an important crop in Haryana with a 30115 ha area and production 643548 MT [1].
Having a healthy and disease-free nursery is the foremost inevitability in determining pro table crop production. Soil inhabitant fungal phytopathogens is a major challenge in the nursery raising. Species of Pythium, Phytophthora, Rhizoctonia, Fusarium, etc cause various soil-borne diseases such as damping off, root rot, wilt, etc [2,3,4]. Damping-off is a global problem of seedlings in the nursery, which may cause 5-80% seedling mortality of different crops [5]. Usage of synthetic fungicides had been a preferred approach since long back in managing the soil-borne fungal phytopathogens. However, issues related to human and soil health, environmental hazards, and disturbed ecosystem [6,7,8,9] pressurized the agriculturists to switch over the plant protection measures towards the safer and eco-friendly approaches [10]. Biological control agents could be an ideal approach for diseases management [11,12,13] as they have no chance to get into the food chain and hence safe to human beings, animals, and plants [14].
Trichoderma is the most widely applied biological tool in plant protection and prospective to minimize the usage of synthetic fungicides in the agriculture sector [15]. It naturally presents in a variety of soils, decaying roots, litter, wood, bark, and other plant materials [ 16,17]. It alone contributes up to ninety percent of total antagonistic fungi [18] and has positive effects on the plants [19] and e cient to take care of diseases of agricultural, horticultural, ornamental crops, etc [20,21] caused by phytopathogenic fungi [22], bacteria, and viruses [23] through various modes of actions [24]. It produces several enzymes for the degradation of the cell wall and numerous volatile metabolites [25,26] for the control of phytopathogens. As of date, about 180 secondary metabolites (antibiotics) are characterized which inhibit or impose a lethal effect on the phytopathogens [27,28]. Its rhizospheric application strengthens the plants to ght against aerial fungal, bacterial, and viral diseases [18] due to elicitors production and resistance induction [29]. T. viride, T. harzianum, T. longibrachiatum, and T. koningii produce volatile antibiotics [30,31]. This antagonist creates unfavorable conditions for the phytopathogenic fungi through acidi cation of the environment [18].
It stimulates plant growth, persuades yield, vitamin production, boosts up nitrogen, and phosphorus availability as well as nutrients mobilization [32]. The antagonist produces zeaxanthin and gibberellins to excel in the seed germination. It also produces different acids to enhance the availability of phosphorus and other micronutrients to the plants [29]. It stimulates the local and systemic resistance in plants [33,34,35,36].
It enhanced the concentration of enzymes and other important proteins for plant resistance development [37,38]. Indigenous isolates of concerned regions performed better in that particular region [33].
T. harzianum and T. virens found potential in managing the pink rot of potato and stem rot of tomato, boosted up potato tuber yield and survival of tomato seedlings [40]. Tomato, lettuce, pepper, bean, and tobacco showed resistance against B. cinerea owing to the application of T. harzianum [41]. It also induced the defense system in cucumber plants [38]. It can be used for soil application [42], seed treatment [43] mixing with farmyard manure [44], and vermicompost. It is capable of controlling the seed and soil-borne diseases of vegetables in the nursery [45]. Its 254 species are documented in the literature [46]; nevertheless, a few species have scope for control of phytopathogens [47]. Though T. asperellum is effective against numerous phytopathogens [48,49] however, comparatively lesser attention has been paid for its agricultural application [50,51].
Integration of Trichoderma sp in plant protection strategies could reduce the dose of synthetic fungicides and improve plant health [52] which is environment friendly as well as cost-effective [53]. Keeping into consideration the disease managing potency and plant growth promotional activity of Trichoderma spp, the present study was planned to test its bioe cacy against the damping-off of the tomato seedlings caused by Fusarium sp in the nursery.

Results
The investigation revealed that the liquid formulation of the antagonist could improve seed germination as well as reduce the damping-off in comparison with control in the nursery when it was applied in different ways, however, soil application, enriched vermicompost, and FYM found more promising.
In the rst year, the seed germinated from 63.88 to 88.75 as compared to 60.13 percent in control and the seedling showed 10.93 to 20.38 percent damping off which was less than the control (26.98%) as indicated in Table 2. Likewise, in the second year, the seed was germinated from 70.83 to 89.50 percent when the antagonist was applied in various ways. The seedlings suffered from damping off disease lesser (8.38-15.33%) as compared to the control bed (20.21%) as evident from Table 3  During 2019 -20, the maximum and minimum temperatures ranged from 11.9 to 22.8 and 2.6 to 8.3 0 C. Relative humidity during the morning was recorded 88.0 to 100 and during the evening it was 46.0-82.0 percent (Fig. 6).

Discussion
Trichoderma spp found effective in controlling the Fusarium wilt of tomato, watermelon, and muskmelon in the greenhouse and eld [54]. Sowing of tomato seeds in T harzianum amended soil led a signi cant decrease in the incidence of Fusarium crown and root rot disease caused by F. oxysporum f. sp. radicislycopersici [55]. Researchers also assessed this antagonist for the control of this disease of vegetable crops through various application methods and noted similar ndings [45].
Seed treatment, seedbed treatment, and enrichment of farmyard manure with T. viride and T. harzianum could offer a better damping-off solution of cauli ower under eld conditions and improved seedling vegetative growth and health [56]. Such treatments were found effective in terms of reduced disease and improved seedling strength and health of tomato and chilli plants [57]. Tomato seed treatment with T. asperellum taken care of P. aphanidermatum [58]. Seed treatment with a consortium of T. harzianum signi cantly declined the damping-off incidence and enhanced the shoot length, chlorophyll content, and yield [59]. Inoculation of tomato seed and nursery soil with T. harzianum spore suspension laid increment in the shoot as well as root length, diameter, and weight when compared to the control. There was a noticeable increase in leaf number, leaf area, chlorophyll content [60].
Application of T. asperellum in eld conditions, managed phytopathogenic Fusarium sp, and signi cantly increased the vegetative growth in respect of root length, shoot length, plant weight as well as chlorophyll content. It also improved total phenol, peroxidase, polyphenoloxidase, and phenylalanine ammonium lyase activity which developed plants resistance against the pathogen [61]. Pre-sowing application of T. viride (WP) in nursery beds of tomato showed a reduction in disease incidence. Nevertheless, growth parameters showed a positive impact [62]. Soil application of T. viride e ciently controlled the pre-and post-emergence damping-off of tomato (P. aphanidermatum) and improved vegetative growth of the plant [63]. T. harzianum and T. virens treated tomato seed revealed up to cent percent seed germination and increased root and shoot length [64].
Trichoderma amended compost could effectively suppress root disease causal fungal phytopathogens viz., F. oxysporum, P. debaryanum, P. aphanidermatum, R. solani in tomato and persuaded seed germination, seedling height, and overall biomass of seedling [65]. T. harzianum enriched organic matter could reduce the tomato damping-off intensity and increase seedling growth [66]. Concurrent application of T. harzianum as seed, seedbed, and soil treatment performed better than its soil application in terms of disease management and better growth parameters of eggplant and tomato. Vermicompost enriched with antagonist increased seed germination, promoted seedling vegetative growth, and minimized damping-off incidence [67]. The disease control and plant growth-promoting property of T. asperellum were established against R. solani in cucumber plant [68]. Inoculation of Trichoderma in tomato nursery and eld could improve plant health in terms of increased the shoot weight, fruit size, yield [69]. Furrow application of T. harzianum in the eld conditions could reduce black scurf incidence in organically grown potatoes [70]. A few Trichoderma strains showed good control of P. ultimum in pea and plant growth promotional property in terms of increased shoot weight, root weight, root length number of lateral roots [71].

Experimental location
The nursery trials were carried out at the research farm at google map position: 29

Preparation of mycofungicide formulation
The liquid formulation (10% Aqueous Suspension) of T. asperellum (KBN-29, accession number ITCC-7764) was prepared following a slightly modi ed method [72,58]. The pure culture of the antagonist was inoculated into potato dextrose agar (HiMedia India Pvt. Ltd.) plates, followed by proper sealing with para lm under aseptic conditions and incubated at 26±2 0 C for 5 days in a BOD incubator. Ten milliliters double-distilled sterilized water was added to each plate and fungal biomass (conidia and mycelia) was scrapped with the help of scrapper. The harvested biomass was further diluted with water and a conidial concentration of approximately 2x10 9 per ml was determined through serial dilution plate inoculation technique (Fig. 1). The prepared formulation was kept in the refrigerator and used within a month.

Source of plants
The locally cultivated tomato var. Selection-7, released by the Department of Vegetable Science, Chaudhary Charan Singh Haryana Agricultural University was used for experimentation. Seed of this variety was obtained from the Vegetable seed store of this Department.

Assessment of different application methods against damping off
The developed formulation was assessed as a seed treatment, soil application, drenching, enriched FYM, and vermicompost. The antagonist treated seed was dried under shade. In soil application, the formulation was sprayed uniformly in to plot before 4-5 hr. It was mixed with FYM and vermicompost before 15 days of its eld application and kept covered under shade. Carbendazim was taken as a standard check in the experiment. There were seven treatments in three replications (Table 1). Raised seedbeds of 90 cm x 45 cm dimensions were prepared in mid-December, 2018, and 2019 (Fig. 2). Two hundred tomato seeds per treatment were sown manually. After 15 days damping-off incidence was recorded. Then drenching of antagonist and carbendazim was done in respective treatments and again after two weeks, disease observation was recorded in comparison with control. After about 45 days of nursery raising, plant growth in terms of shoot and root length (cm) was measured from ten randomly sampled seedlings per treatment.

Data analysis
The experimental data were statistically analyzed through the online package 'OPSTAT' of the institute.
Weather conditions during the study period Weather data of temperature, relative humidity, wind speed, sunshine hours, and rainfall were collected from the Department of Agrometeorology, College of Agriculture, Chaudhary Charan Singh Haryana Agricultural University, Hisar.

Conclusion
The present investigation showed that the tested antagonist's formulation could e ciently managed the tomato damping-off as well as encouraged the vegetative growth of seedlings which ultimately ensured Page 7/19 better and healthy seedling. And this formulation can successefully used through different methods to take care of tomato damping off.

Consent for publication
The authors agree to publish this paper. The data has not been published partially or completely in any other journal.

Availability of data and materials
The datasets generated and analyzed during the current study are not publicly available due to privacy issues but are available from the corresponding author on reasonable request.

Competing interests
The authors declare that there is no con ict of interest regarding the publication of this paper. It is declared that the authors have no competing interests.

Funding
There is no nancial support for this purpose from any funding agency at this time; it was carried out as an in-house study. However, the BMC (waivers) Plant Biology Journal considered for review process without processing charge.
Kishor Chand Kumhar generated the idea of the study, prepared mycofungicide formulation, decided treatments, recorded disease incidence, analyzed, and presented data in tables and graphs. Kuldeep Kumar arranged seed, raised nursery, and recorded data on growth parameters. Indu Arora has taken care of the nursery during the study period. Arun Kumar Bhatia and Vinod Kumar Batra timely monitored and suggested the required follow up action.    Figure 1 Steps   Weather condictions during second year (2019-20)