Introduction

Tomato is an indispensable vegetable crop which is the major source of nutrients and medicinal values, hence known as ‘Nutraceutical vegetable’ (Singh et al. 2019). Tomato is highly adaptive to warm season and can be grown successfully in plains as well as in hills. Cultivation of tomato in rainy season is assuming a great importance in the north-eastern region of India in general and Nagaland in particular owing to its high prices of produce obtained from other parts of the country during this period (Babu 2006).

Though tomato crop occupies a very important place among the vegetable crops cultivated in India, the average yield of this crop on farmers’ fields is reasonably poor. One of the constrain for poor yield is the devastating effect of certain diseases. Among the diseases, late blight of tomato caused by Phytophthora infestans is destructive and wide spread in nature (Son et al. 2008). Worldwide losses were estimated is about $170 billion annually and thus this pathogen was considered as a major threat for global food security (Latijnhouwers et al. 2004; Wu et al. 2012). Yield losses up to 79% from late blight damage in tomato have been recorded in India (Arora et al. 2014; Chowdappa et al. 2015).

Although, several management components viz., cultural practices, biological agents, host resistance and fungicides are available, but fungicides hold promise in managing the late blight disease of tomato. Use of fungicides is costly, may lead to environmental pollution and less effective due to increasing resistance of the pathogen. Under such conditions, the most effective method is the biological control (Harish et al. 2008). In recent years, emerging strategy is integrated biological control as microbial consortia. Under field conditions microbial consortia are much more efficient than single strain of organisms with diverse metabolic capabilities (Yan et al. 2002).

Hence, looking into the aforesaid realities, the use of native biological agents as a consortium and also not much systematic research work has been carried out on late blight disease of tomato under Nagaland condition. Hence, in the present study attempts were made to explore native isolates of BCAs and developing an indigenous microbial consortium package for developing a biointensive management strategy against late blight disease of tomato along with yield enhancement.

Materials and methods

Identification of the pathogen

The causal agent of late blight disease of tomato was isolated by standard tissue isolation technique on rye-A agar medium (Hollomon 1965). The purified isolate was subjected to pathogenicity test. For this purpose, isolated pathogen was inoculated on 4 weeks old susceptible tomato cv. Pusa Ruby (Loliam et al. 2012).

Morphological characters of the pathogen was studied on host as well as in pure culture on rye-B agar medium. The isolated pathogen was identified on the basis of morphological characters as documented by Waterhouse (1963).

Isolation and identification of bioagents

A field survey was undertaken for the collection of rhizospheric soil samples from different cropping areas in Nagaland, India (Table 1). Soil samples were taken from the rhizosphere of healthy plants and kept in polyethylene bags. The individual sample was mixed thoroughly after air drying. Thirty three isolates were obtained from the collected samples by soil dilution plate technique (Waksman 1927).

Table 1 Native biocontrol agents (BCAs) and their collection locations
Full size table

Initially isolated microbes were identified as Trichoderma spp. and Pseudomonas spp. based on morphological characteristics by use of selective media viz., Trichoderma selective medium (TSM) (Elad and Chet 1983) and King’s B medium (King et al. 1954) respectively. Further, the potential isolates were identified as T. asperellum (T-11; Acc. No MK928414 and T-14; Acc. No MK928417) and P. fluorescens (Pf-2; Acc. No MN783298 and Pf-3; Acc. No MN783297) based on the molecular characterization (Singh et al. 2020).

In-vitro antagonistic tests

The antagonistic effect of Trichoderma and Pseudomonas isolates were evaluated against Phytophthora infestans by dual culture plate technique as per Sivakumar et al. (2000) and Georgakopoulos et al. (2002) respectively. Linear mycelial growth of the pathogen was recorded in Petri plate after full growth of pathogen attained in control treatment. The per cent inhibition of the growth of pathogen by antagonists over control was calculated (Vincent 1927).

Investigation on the biocontrol mechanisms of antagonists

The effects of volatile metabolites and mycoparasitism activity of isolated BCAs were assessed against P. infestans by adopting the technique given by Dennis and Webster (1971) and Rodrigues (2010), respectively. The production of Ammonia, IAA and HCN by Trichoderma and Pseudomonas isolates were also determined in the qualitative assay technique given by Cappuccino and Sherman (1992), Gordon and Weber (1951) and Miller and Higgins (1970), respectively. Phosphate solubilization and siderophore production test was also conducted qualitatively by inoculation of Trichoderma and Pseudomonas isolates on National Botanical Research Institute’s phosphate (NBRIP) agar medium (Nautiyal 1999) and chrome azurol sulfonate (CAS) agar medium (Milagres et al. 1999), respectively.

Selection of potential isolates and their compatibility study in-vitro

Based on in-vitro antagonistic capabilities of Trichoderma and Pseudomonas isolates against P. infestans and elucidation of their various biocontrol mechanisms, the potent isolates were selected for further studies. In-vitro compatibility test amongst microbial consortia of potent isolates of Trichoderma and Pseudomonas were evaluated by dual culture plate method (Siddiqui and Shaukat 2003) in order to determine the compatibility among different combination of consortia.

Antagonistic efficacy of microbial consortia against P. infestans

The in-vitro bioassay technique was used for the testing of microbial consortia against P. infestans. The mycelial disc (10 mm diameter) of the pathogen (9 days old) was placed at centre of Petri plate containing rye-B agar medium (20 ml). Simultaneously, 10 mm diameter disc of potent Trichoderma (T-11 and T-14) isolates (9 days old) and 20 µl of an overnight culture of potent Pseudomonas (Pf-2 and Pf-3) isolates were poured in wells (5 mm diameter) at different corner of Petri plate. Linear mycelial growth of the pathogen was recorded in Petri plate when mycelium of test pathogen touched any antagonists in any treatment. The per cent inhibition of the growth of pathogen by antagonists over control was calculated (Vincent 1927).

Preparation of liquid based bio-formulation of microbial consortia

The conidial suspension of each selected isolates of T. asperellum (T-11 and T-14) was prepared from 9 days old PDA plates. The plates were rinsed with sterile distilled water and the mycelia were carefully scraped off with a bent glass rod. This suspension was filtered through filter paper (Whatman No.1) to separate the spores from the mycelia. The spore concentration was adjusted to 3.7 × 108 spores/ml (Dubos 1987) with the help of haemocytometer. Similarly, selected P. fluorescens isolates (Pf-2 and Pf-3) cell suspension was prepared by inoculating into King’s B broth followed by shaking for 48 h (150 rpm) at 28 °C. The bacterial suspension was adjusted optically at 1 × 109 cfu/ml (Mulya et al. 1996). Liquid based bio-formulations of consortia were prepared by mixing equal volume of each selected isolate just before use for field experiment (Srinivasan and Mathivanan 2009).

Field evaluation of liquid bio-formulation of microbial consortia against late blight of tomato under natural epiphytotic conditions

The field trials were conducted during the tomato growing seasons (Sept.–Jan.) of 2017–2018 and 2018–2019. The research field site is located in the foothills of Nagaland (India) and situated at 25° 45′ 45″ North latitude and 93° 51′ 45″ East longitudes at an elevation of 310 m above mean sea level.

The bio-formulation of microbial consortia (MC) and chemical treated seeds (400 seeds/treatment) of tomato were sown in nursery beds (8 × 1 cm at 1 cm depth) after 15 days of formalin (2%) treated soil. The tomato cv. Pusa Ruby was used in the field experiment, which is known to be highly susceptible to P. infestans in India (Singh et al. 2019). The 28 days old seedlings were transplanted (60 × 45 cm) in main field during second week of October in raised plot (1.8 m × 1.8 m). The each plot was framed at 50 cm distance apart. All the recommended standard cultural operations were followed.

The field experiment was laid out in a randomized block design (RBD) with six replications (72 plants per treatment). A total of four treatments viz., T1 (MC-1; seed treatment (1%) + soil application (1%) + foliar sprays (1%) at 15, 30 and 45 DAT), T2 (MC-2; seed treatment (1%) + soil application (1%) + foliar sprays (1%) at 15, 30 and 45 DAT), T3 (Chemical control; seed dressing with 0.3% Captan 50% WP + soil application of Mancozeb 75% WP (0.2%) + foliar sprays of Ridomil MZ 72% WP (0.25%) at 15, 30 and 45 DAT) and T4 (Control, sterile distilled water) were used.

Application methods of liquid microbial consortia

Seed treatment

The surface sterilized (1.0% sodium hypochlorite for 2 min) seeds were soaked in conidial suspension of microbial consortia at 1%, chemical control treatment (0.3% of captan 50% WP) and control treatment (soaked in sterile distilled water). All the treated seeds were dried by keeping under aseptic condition in laminar air flow for 5 h (Srinivasan and Mathivanan 2009).

Soil application

The soil application treatment was done with 1% of MC inoculated in FYM, Mancozeb 75% WP at 0.2% and sterile distilled water for control treatment at 10 days before transplanting (Srinivasan and Mathivanan 2011).

Foliar spray

Three foliar sprays were done with 1% of MC, Mancozeb + Metalaxyl-72% WP at 0.25% and sterile distilled water for control treatment at 15, 30 and 45 DAT. The total spray solution of 150 ml was used in each plot (12 plants) (Srinivasan et al. 2009).

Observations

The late blight disease severity was assessed visually on leaves, stems and fruits of all plants in each replication following rating scale as per Irzhansky and Cohen (2006), when all plants in control treatment infested with late blight disease under natural epiphytotic conditions. The severity grades were converted into percentage disease index (PDI) for analysis as per the formula given by Wheeler (1969).

Plant growth promoting attributes like plant height, number of leaves, number of branches, number of fruits per plant, fresh weight of fruit, marketable fruit yield and root length were recorded.

Statistical analysis

The data were analyzed using WASP 2.0 software developed by the Central Coastal Agricultural Research Institute, Goa (India).

Results

Identification of the pathogen

The pure culture was obtained from the diseased specimens were identified as P. infestans based on macroscopic and microscopic characters. The phenotypic characteristics of isolate were observed fluffy cottony mycelium and slow growth rate on the rye-B agar medium. Microscopic observation revealed that the fungal hyphae were hyaline, moderately thick hyphae, coenocytic and profusely branched. Sporangiosphores were sympodial with a small swelling at the base of each branch. Sporangia were terminal or lateral, ellipsoid, ovoid or limoniform, semipapillate, deciduous and pedicelless and they comparatively more frequently observed on the tomato plants than in pure culture. Chlamydospores of the pathogen were also recorded in diseased specimens (Fig. 1).

Fig. 1
figure 1

Characterization of the pathogen (P. infestans)

Full size image

In-vitro antagonistic tests

Altogether 25 isolates of Trichoderma were screened for their inhibitory action on the radial growth of P. infestans. It was found that the growth of the pathogen in dual culture plates progressed until they come in contact with the leading edges of the antagonist. The per cent inhibition over control was calculated and it was observed that T-11 was the most promising isolate against P. infestans with 73.73 per cent inhibition. Next best isolate was T-14 (66.67%) followed by T-5 (64.93%), T-25 (64.00%) and the least antagonistic effect was observed in T-17 (51.07%) at 8 days after incubation at 18 ± 1 °C (Table 2).

Table 2 In-vitro screening of native biocontrol agents (BCAs) against P. infestans and their mechanisms
Full size table

The antagonistic effects of Pseudomonas isolates were evaluated against P. infestans which significantly inhibited the growth of the pathogen as compared to control treatment. Among the Pseudomonas isolates, maximum per cent inhibition was observed in Pf-2 (81.33%) which is significantly superior to all other treatments followed by Pf-3 (73.33%), Pf-1 (69.33%), Pf-7 (66.67%) and Pf-4 (65.33%) at 8 days after incubation at 18 ± 1 °C (Table 2). The clear zone of inhibition was also observed in the dual culture plate of Pf-2 and Pf-3.

Investigation on the biocontrol mechanisms of antagonists

The effects of volatile metabolites of Trichoderma and Pseudomonas isolates were assessed against P. infestans. Among the tested isolates, the per cent inhibition over control was calculated and it was recorded that Trichoderma (T-11) and Pseudomonas (Pf-3) was found to be most promising in production of volatile compounds against P. infestans with 45.55 and 53.67 per cent inhibition (Table 2). The mycoparasitism activity of 25 isolates of Trichoderma were also assessed against P. infestans and they showed the presence of coiling as hyphal interactions between them (Table 2).

The production of Ammonia by Trichoderma and Pseudomonas isolates were also determined in the qualitative assay. Among the tested isolates, Pseudomonas isolates (Pf-3, Pf-4, Pf-7 and Pf-8) and Trichoderma isolates (T-1, T-2, T-11, T-14 and T-25) exhibited strong ammonia production by turning initial peptone water broth from yellow to dark brown colour (Table 2). The results of qualitative assay of IAA production by different native BCAs revealed that Pseudomonas isolates (Pf-2 and Pf-3) and Trichoderma isolates (T-11 and T-14) exhibited strong IAA production. The moderate production of HCN was observed in Pseudomonas isolates (Pf-2, Pf-3 and Pf-8) (Table 2).

The results of qualitative assay of phosphate solubilization by different native BCAs revealed that Pseudomonas isolates (Pf-2, Pf-3 and Pf-8) and Trichoderma isolates (T-11 and T-14) elucidated strong phosphate solubility activity (Table 2). The siderophore production test was also conducted qualitatively by inoculation of Trichoderma and Pseudomonas isolates on chrome azurol sulfonate (CAS) agar medium. All 33 isolates showed positive results for siderophore production. Among the tested isolates, Pseudomonas isolates (Pf-3 and Pf-8) and Trichoderma isolates (T-3, T-4, T-5, T-7, T-8, T-9, T-10, T-11, T-14, T-15, T-18 and T-21) exhibited strong siderophore production by pink and orange halo colour development (Table 2).

Selection of potential isolates and their compatibility study in-vitro

Based on in-vitro antagonistic capabilities of Trichoderma and Pseudomonas isolates against P. infestans and elucidation of their various biocontrol mechanisms, the potent isolates of Pseudomonas (Pf-2 and Pf-3) and Trichoderma (T-11 and T-14) were used for further studies. Selected native microbial isolates were showed consistent ability to produce siderophore, ammonia, IAA, volatile metabolites and also able to release inorganic phosphorus from tri-calcium phosphate (Table 2).

In-vitro experiment was carried out in all permutations and combination amongst the potent isolates of Trichoderma and Pseudomonas. Altogether 11 treatment combinations were tested and compared with growth of Pf-2 (Control-1), Pf-3 (Control-2), T-11 (Control-3) and T-14 (Control-4). The microorganisms showing positive compatibility among them was recorded, tabulated and selected for further study. The data showed compatibility among all the treatment combinations of the four bioactive microorganisms in-vitro. No clear inhibition zone was recorded between the tested microbial consortia. Absence of inhibition zone indicated that the potential isolates of Trichoderma and Pseudomonas were compatible with each other.

Antagonistic efficacy of microbial consortia against P. infestans

A total of 12 treatment combinations were compared. Eleven consortia produced varying inhibitions (%) in-vitro against P. infestans (Table 3 and Fig. 2). All consortia tested against P. infestans were significantly superior over control. Among the different consortial sets tested in-vitro the significant highest inhibition of pathogen was recorded in the combination of Pf-2 + Pf-3 + T-11 + T-14 (83.33%) followed by Pf-2 + Pf-3 + T-11 (78.38%), Pf-2 + Pf-3 (77.43%) and Pf-2 + T-14 (76.54%) respectively at 5 days after incubation at 18 ± 1 °C.

Table 3 In-vitro antagonistic effect of microbial consortia on radial growth and per cent inhibition of P. infestans
Full size table
Fig. 2
figure 2

In-vitro antagonistic effect of microbial consortia (MC) on radial growth of P. infestans

Full size image

An in-vitro study was taken up to select the two best microbial consortia (MC) against the test pathogen. The MC-1 (P. fluorescens Pf-2 + P. fluorescens Pf-3 + T. asperellum T-11 + T. asperellum T-14) and MC-2 (P. fluorescens Pf-2 + P. fluorescens Pf-3 + T. asperellum T-11) inhibited the pathogen significantly and were found to be the most effective consortia. Hence, they were selected for field study.

Field evaluation of liquid bio-formulation of microbial consortia against late blight of tomato under natural epiphytotic conditions

Among different treatments, liquid microbial consortia (MC)-1 significantly decreased the late blight severity (12.08 PDI) compared to all other treatments. This was comparable with the chemical treatment (6.25 PDI). In case of untreated control, high severity of 77.36 PDI was recorded at 45 DAT (Table 4). These results revealed that the chemical control significantly decreased late blight severity (91.92%) over control treatment. Next in order of merit was MC-1 (84.38%) and MC-2 (77.20%). The mortality per cent significantly decreased in all the treatment (100%) over control treatment (Table 4). Simultaneously, liquid bio-formulation of MC-1 significantly increased the tomato plant height (101.20% and Fig. 3), number of leaves per plant (116.48%), number of branches per plant (146.57%), number of fruits per plant (185.52%), fresh weight of fruit (42.59%), root length (67.28%) and marketable fruit yield (313.02%) over untreated control (Tables 5 and 6).

Table 4 In-vivo effects of microbial consortia (MC) on per cent decrease of late blight severity and mortality per cent and per cent increase of marketable tomato fruit yield over control
Full size table
Fig. 3
figure 3

Effect of microbial consortia (MC) on tomato plant height, number of leaves, fruits and branches; T1 (MC-1), T2 (MC-2), T3 (Chemical control) and T4 (Control)

Full size image
Table 5 In-vivo effects of microbial consortia (MC) on tomato plant height, number of leaves and branches per plant at 94 DAT
Full size table
Table 6 In-vivo effects of microbial consortia (MC) on number of tomato fruit per plant, fresh weight of fruit and root length
Full size table

Discussions

The pathogen was identified as P. infestans based on the nature of disease observed, morphological and cultural characters seen under the microscope. These characters were further compared with the characters reported by Waterhouse (1963) and description given by Agrios (1997) and Zentmyer (1983). The present observations corroborates with the descriptions and findings of earlier workers.

The development of native bio-formulation is more efficient antagonistic player in plant disease management and growth promotion. Antagonistic effect of Trichoderma and Pseudomonas isolates against P. infestans has already been reported by several research workers (Kabir et al. 2013; Kumar et al. 2015; Lamsal et al. 2013; Patel and Mukadam 2011; Zegeye et al. 2011). In this study, the probable reasons of high inhibitory activity of the antagonists observed on P. infestans in dual cultures may be due to production of antifungal metabolites such as mycoparasitic activities, volatile gases, cell-wall degrading enzymes, HCN, siderophores, pyoluteorin, pyrrolnitrin and 2-4 diacetyl phloroglucinol.

Many secondary metabolites have been recorded to be involved in microbial interactions (Dennis and Webster 1971; Kapri and Tewari 2010; Vespermann et al. 2007). These reports are commensurate with result of the present investigation, which suggests that the production of secondary metabolites by both Trichoderma and Pseudomonas isolates have definite influence on the high degree of inhibition of P. infestans.

The mycoparasitic potential of Trichoderma spp. against P. infestans is well documented in previous findings (Ezziyyani et al. 2007; Pugeg and Ian 2006; Zegeye et al. 2011). However, in the present investigation, working with the native isolates, the mycoparasitic potential was further manifested by characteristic envelopment and coiling around the hyphae by all isolates of Trichoderma spp. The hyphae of Trichoderma spp. was also observed to grow in close proximity to the hyphae of P. infestans before coagulation and disintegration occurred.

Dixit et al. (2015) evaluated 11 isolates of fluorescent Pseudomonas for ammonia production. All isolates showed positive result for ammonia production. Lalngaihawmi and Bhattacharyya (2019) also evaluated Trichoderma spp. for ammonia production and results revealed that all the Trichoderma spp. showed positive result. These reports are in agreement with the result of the present investigation, which suggests that the production of ammonia by both Trichoderma and Pseudomonas isolates have positive impact on the plant growth.

Dixit et al. (2015) further evaluated 20 isolates of Trichoderma for IAA production. All Trichoderma spp. isolates elucidated positive results for IAA production. Prasad et al. (2017) also evaluated 24 isolates of Trichoderma spp. and 12 isolates of B. subtilis and P. fluorescens for IAA production. In the present investigation, 3 isolate of Pseudomonas and 12 isolates of Trichoderma were observed to produce IAA at varying intensity. This occurrence may be ascribed to the heterogeneous nature of the source and the strains of the antagonists.

Corbett (1974) described that HCN inhibits proper functioning of enzymes and natural receptors reversible mechanism of inhibition in the pathogens. This report is in agreement with the result of the present investigation, which suggests that the production of HCN by Pseudomonas isolates have absolute influence on the high degree of inhibition of P. infestans.

It has been observed by many investigators (Bhakthavatchalu et al. 2013; Gangwar et al. 2012; Kapri and Tewari 2010; Prasad et al. 2017; Rai 2017) that a high proportion of phosphate solubilizing microorganisms (PSMs) reside in the rhizosphere of plants and play an important role in solubilization of bound phosphates, making them available to the plants. This report is in agreement with the result of the present investigation, which suggests that the phosphate solubilization by both Trichoderma and Pseudomonas isolates have obvious influence on the plant growth.

In this present study, the strong and positive siderophore production exhibited by Pseudomonas isolates (Pf-3 and Pf-8) and Trichoderma isolates (T-3, T-4, T-5, T-7, T-8, T-9, T-10, T-11, T-14, T-15, T-18 and T-21) explicate the corresponding inhibited radial growth and high per cent inhibition of P. infestans.

Microbial consortia are known to enhance plant growth, which can result in development of various plant parts and higher growth leads to significant enhancement of vegetative growth attributes through plant growth promotion, whereas growth promotion was absent in chemical control in addition to disease suppression. Presence of consortia in the rhizoshphere increases the availability of nutrients through solubilization of insoluble sparingly soluble minerals have better nutrient uptake thereby enhancing plant growth (Biam and Majumder 2019; Harish et al. 2008; Idris et al. 2007; Raupach and Kloepper 1998; Yan et al. 2002).

Based on activities of biological control mechanism and plant growth promotion studies, the best microbial consortium was identified as MC-1. This promising indigenous liquid consortium has promoted the tomato plant growth and reduced the losses due to late blight disease in an eco-friendly manner exhibiting tremendous potential for its commercial exploitation.