Role of plant growth promoting microbes in the control of fungal foliar diseases of tomato under protected cultivation

This study was conducted to assess the effectiveness of plant growth-promoting microbes on the foliar disease of organic tomatoes under protected cultivation. The biological control agents, viz., Trichoderma harzianum (T. harzianum), Bacillus subtilis (B. subtilis), and Pseudomonas fluorescens (P. fluorescens), used seed treatment, seedling dip, and spray separately in sequence with the plot size of (2.5 × 1 m) and the spacing of (45 × 60 cm). The observations on percentage disease yield and plant growth parameters were assessed, and economics was calculated. Tukey's HSD at p < 0.05 was used to compare all means among treatments. The results showed that seed treatment with B. subtilis at 5 g, followed by a seedling dip with B. subtilis at 10 g l−1 and spray with B. subtilis at 5 g l−1 four times at 15 days intervals significantly (p < 0.05) reduced the tomato diseases early blight, septoria leaf spot, and powdery mildew (T2). These results were comparable to seed treated with P. fluorescens at 5 g l−1, followed by a seedling dipping with P. fluorescens at 10 g l−1 and spraying with P. fluorescens at 10 g l−1 four times at 15-day intervals (T3). Except for T1, plant parameters, yields, net returns, and benefits were significantly higher (p < 0.05) than the untreated control (T8). The applications of B. subtilis and P. fluorescens as plant growth-promoting microbes had a considerable scope for managing fungal foliar (multiple) diseases and achieving sustainable yields under the protected cultivation of organically grown tomatoes.


Background
Tomato cultivation in open fields is challenging throughout the year due to unpredictable weather conditions and plant diseases that affect the development and quality of the produce (Arushi and Banyal 2018). Tomatoes grown in protected settings produce larger yields, extend the harvest season, improve quality, and increase production stability, resulting in peak performance and reliable off-season supply (Pooja and Hakkim 2017). The protected cultivation of crops enhances the success of biological control agents with greater precision than the field crops. Increased education and extension of proven, ready-to-use biological control options are vital strategies to accelerate adoption (Atalla et al. 2020). There are reports (Kabdwal et al. 2019) of tomato loss in the field health risks associated with agrochemicals and how these issues could be addressed by adopting organic protected cultivation. There exists scope for growing organic vegetables under protected structures. Tomatoes are affected by many plant pathogens, such as bacterial, fungal, nematode, viral diseases, and abiotic factors. Among the most prevalent fungal foliar infections, early blight, septoria leaf spot, gray leaf spot, and powdery mildew are the ones that cause havoc across growing regions globally (Sokhi Page 2 of 10 Hegde et al. Egyptian Journal of Biological Pest Control (2022) 32:105 et al. 1991). The cultivation of tomatoes using biocontrol agents was very effective in developing environmentally safe, long-lasting, and effective I.P.M. programs to manage tomato foliar diseases (Kaur et al. 2016). Tomato cultivation in a polyhouse is a well-known technology, and so is the organic production of tomatoes. However, most studies were carried out separately, either as organic production of tomatoes or tomatoes grown under protected structures. While our examinations conducted during 2017-2018 and 2018-2019 was the combination of organic cultivation of tomato under polyhouse conditions, which draws the attention of growers who wishes to practice organic tomato production under polyhouse conditions. The sequential use of microbes alone to control multiple fungal diseases of tomatoes requires the attention of growers and biopesticide industries. In the era of organic agriculture, biopesticides have a promising role in managing various diseases and influencing plant growth-promoting parameters. Thus, growing pesticide-free tomatoes under polyhouse conditions will be a bio-based technology in augmenting farmers' income and benefiting plant, soil, and human health. This study was conducted to assess the effectiveness of plant growth-promoting microbes on the foliar disease of organic tomatoes under protected cultivation.

Methods
A polyhouse experiment was carried out at the Institute of Organic Farming Bio-Resource Farm, Agricultural Sciences University, Dharwad, for two consecutive years, 2018 and 2019. The experimental site was an organically certified farm, and soil fertility was maintained by the application of Farmyard manure (350 kg acre −1 ), vermicompost (200 kg acre −1 ), and Neem cake (100 kg acre −1 ) based on soil test results.

Seed treatment (ST), seedbed preparation, and sowing
Moderately susceptible indeterminate tomato hybrid Sarpan-8 seed was taken for the study. The talc-based formulations of B.C.A.s were used for the present study. The seeds treated with B.C.A.s @ 5 g kg −1 were sown to a depth of 0.5 cm in the primary seedbed. Seeds were germinated in 4-6 days, and the seedlings were ready for transplanting by 25 days after sowing. Seedlings were dipped in various solutions of Trichoderma harzianum (T. harzianum), Bacillus subtilis (B. subtilis), and Pseudomonas fluorescens (P. fluorescens) Trichoderma harzianum (T. harzianum), Bacillus subtilis (B. subtilis), and Pseudomonas fluorescens (P. fluorescens) @ 10 g l −1 of water and shade dried for 20 min before transplanting. The soil area inside the polyhouse was thoroughly dug to 30 cm. All the weeds, stones, stubbles, etc., were removed entirely. The soil was irrigated three to four times, ploughed again, and brought to a fine tilth. Raised beds of 30 cm in height and 100 cm in width were prepared with a walking space of 60 cm among beds. The beds were incorporated with F.Y.M. @ 2.5 t ha −1 , vermicompost @ 2.00 t ha −1 , neem cake @ 2.00 t ha −1 , and rock phosphate @1.00 t ha −1. These were added to soil at different intervals of crop growth. Additionally, phosphate solubilizing bacteria (PSB) (100 g −unit ) like T. harzianum (accession no. NAIMCC-F-03328) (2 × 10 6 ) and P. fluorescens (accession no. NAIMCC-B-01981) (2 × 10 6 ), Purpureocillium lilacinum (accession no. IOF Dharwad) at (2 × 10 6 ) and Metarhizium anisopliae (IOF Dharwad) (2 × 10 6 ) @ 2 kg t −1 of organic matter were added to soil at 15 days before transplanting. At the center of the bed, two inline dripper laterals were placed. Inline dripper lateral had an emitting point for every 30 cm interval with a discharge of 2 l hr −1 . Paired row system of planting with a zigzag manner was followed to have more aeration and space among plants.

Assessment of diseases
The diseases like early blight were recorded on a 5-point scale, while powdery mildew and septoria leaf spot were recorded on a 10-point scale as given by Mayee et al. (1985).
Early blight 0-5 scale Small irregular, brown spots covering 1-10% of the leaf area 5 Small lesions, but enlarged with a dark brown margin with a gray center covering 11-25% of the leaf area 7 Enlarged lesions with a dark brown margin with a gray center covering 26-50% of the leaf area 9 Lesions coalesce to form irregular dark brown to black patches covering 51%or more leaf area Percentage disease index (P.D.I.) was calculated by using the formula (Mayee et al. 1985).
The first spray was immediately given after the appearance of early blight and septoria leaf spot. Subsequent sprays were presented at an interval of 15 days. Four sprays were given @10 g l −1 to manage the fungal foliar diseases. The early blight symptoms appeared firstly (30 days after transplanting) and symptoms of powdery mildew and septoria leaf spot disease appeared later (75 days after transplanting). Sulphur was recommended for powdery mildew of tomato and was certified in the organic cultivation of vegetables. Hence it was used as a treated check. An untreated check was maintained for comparison.

Characterization of antagonistic microorganisms used in the study
HCN (hydrogen cyanide) production by Bakker and Schippers (1987), siderophore production by Schwyn and Neilands and chitinase production by Agrawal and Kotasthane (2012) were assessed by using standard protocol with slight modifications. After incubation for a week at 30 °C, HCN production potential was assessed based on colour change of filter paper from deep yellow to orange and finally orange brown to dark brown. Orange halos produced around the colonies indicated the production of siderophores. Colour change from yellow to purple around the colony indicated chitinase production.

Observations on plant, yield, and cost parameters
Observations on plant height, number of fruits per cluster, number of pickings, yield per plot, and cost economics were recorded at 120 days.

Statistical analysis
Statistical analysis was carried out per the standard procedures (Panse and Sukhatme 1954). Data analysis was done using R (R Core Team 2018). The differences among the means were analyzed by two-way ANOVA with mean separation by Tukey's HSD at p < 0.05 to determine whether there were significant differences among the treatments (T1-T8) and their interactions. A significant level was set at the probability level of *p < 0.05; *p < 0.01; ***p < 0.001 for all the analyses.

Septoria leaf spot
The disease incidence during the Kharif 2017-2018 (Table 1), the septoria leafspot incidences ranged from 13.32% (T2) to 14.80% (T8) after the third spray. The lowest septoria leaf spot diseases were recorded in the T2 (23.16%), T3 (26.40%), T5 (24.78%), and T6 (24.12%), compared to control (41.42%) during the Kharif, 2018-2019. The pooled data of both the years also showed that the early blight incidences were significantly lower (p < 0.05) in T2 (21.41%) and T3 (20.66%), and the powdery mildew incidences in T2 (13.82%) and T3 (15.48%) and septoria leaf spot incidences in T2 (19.35%) and T3 (22.08%) had also followed similar trend. The biocontrol agents viz., T. harzianum and B. subtilis had shown positive results for the production of HCN by changing the colour of filter paper that was pre-added with picric acid from light yellow to orange (Fig. 1a). P. fluorescens tested negative for the HCN production as it didn't change the colour of filter paper from yellow to orange. P. fluorescens and B. subtilis produced yellow halos around the colony confirming the siderophore production (Fig. 1b). T. harzianum showed a negative result for the siderophore production as it did not produce yellow halos around the mycelial growth in the CAS (Chrome Azurol S) media. T. harzianum was found to be positive for the test. It was observed by a change in media colour from pinkish-red to purple. P. fluorescens and B. subtilis have shown negative for the chitinase test as the colour of the media did not change (Fig. 1c).

Discussion
This application of biocontrol agents (viz., P. fluorescens, B. subtilis, and T. harzianum) revealed promising effectiveness against tomato fungal foliar disease. It was found that the fungal bioagent, T. harzianum was more effective than the bacterial antagonists P. fluorescens and B. subtilis Page 7 of 10 Hegde et al. Egyptian Journal of Biological Pest Control (2022) 32:105 ( (Basamma and Kulkarni 2017). Promotions of plant growth (more plant height and more fruit per cluster and picking) often demonstrated the effectiveness of applying these bioagents. Net income and benefit: cost ratios were also found maximum in P. fluorescens, B. subtilis, and T. harzianum treated plots (T7) compared to the untreated control (T8). These results agreed with the studies conducted on cucumbers under protected cultivation (Joshi et al. 2010). Many strains of P. fluorescens enhanced plant growth promotion and reduced the severity of various diseases. PGPR (Plant Growth Promoting Rhizobacteria) are highly diverse biocontrol agents, and their effects can occur via local antagonism to soil-borne pathogens or by induction of systemic resistance against pathogens throughout the entire plant (Compant et al. 2005). Among the evaluated biocontrol agents, T. harzianum and B. subtilis showed positive results, for the production of HCN by changing the colour of filter paper from yellow to orange. HCN production by the antagonists was reported by other workers as well (Qaiser et al. 2018). The HCN is known to inhibit electron transport, disrupting the energy supply to the cells, may act as toxic to pathogen and ultimately leads to the death of pathogens thus reducing plant disease (Knowles, 1976). P. fluorescens and B. subtilis showed positive results for the siderophore production by developing yellow halos around the colony in the CAS agar medium. Siderophores are iron-chelating ligands secreted extracellularly by antagonists under iron liming conditions. They increase the solubility of ferric ions (Fe 3+ ), making them available to plants and preventing pathogens from acquiring iron (Loper and Buyer 1991).  Trichoderma harzianum showed positive for the production of chitinase by changing media colour to purple. Chitinase is the enzyme that breaks the cell wall of pathogens, thus suppressing the pathogens. These findings are supported by Khatri et al. 2017. Several substances produced by antagonistic rhizobacteria have been related to pathogen control and indirectly promoted growth in many plants, such as siderophores and antibiotics (Tariq et al. 2017). Rhizobacteria belonging to the genera, Pseudomonas and Bacillus are well known for their antagonistic effects and ability to trigger induced systemic resistance (Jogaiah et al. 2018). After sequential application of P. fluorescens and B. subtilis reduced the disease incidence significantly (p < 0.05), enhanced flowering, and resulted in more pickings. A similar trend was reported in term yield in tomatoes ( Kabdwal et al. 2019). Similarly, BCAs viz., T. harzianum, T. longibrachiatum, T. atroviride have proved potential antagonism against Alternaria solani (Imran et al. 2022) in different combinations in open field trials. Seedling treatment and foliar spray for growth promotion and disease control in tomatoes proved efficacy. A. solani and S. lycopersici causing the tomato early blight and septoria leaf spot under both in-vitro and polyhouse conditions resulted in reduced disease incidence, improved yields, and overall net yields. Usage of plant growth-promoting microbes increased plant height, which helped the plants bear more flowers and produce more fruits.

Conclusions
The present study on the organic cultivation of tomatoes with special regard to the use of PGPRs as biopesticides under protected cultivation showed significant reduction in disease incidence, enhanced flowering, and resulted in more pickings. To manage fungal foliar diseases of tomato in a cost-effective manner, a set of standardized experimental design and seed treatment, followed by sequential spray at 15-day intervals were required.