Ecological performance of multifunctional pesticide tolerant strains of Mesorhizobium sp. in chickpea with recommended pendimethalin, ready-mix of pendimethalin and imazethpyr, carbendazim and chlorpyrifos application

The present study was designed to screen the Mesorhizobium strains (50) for tolerance with four recommended pesticides in chickpea. In-vitro, robust pesticide tolerant strains were developed in pesticides amended media over several generations. Further, verification of the multifunctional traits of pesticide tolerant mesorhizobia under pesticide stress was conducted in-vitro. Among different pesticides, significantly high tolerance in Mesorhizobium strains was observed with recommended doses of pendimethalin (37%) and ready-mix (36%) followed by chlorpyrifos (31%) and carbendazim (30%), on an overall basis. Based on multifunctional traits, Mesorhizobium strains viz. MR2, MR17 and recommended MR33 were the most promising. Ecological performance of the potential Mesorhizobium strains alone and in dual-inoculation with recommended PGP rhizobacterium strain RB-1 (Pseudomonas argenttinensis JX239745.1) was subsequently analyzed in field following standard pesticide application in PBG-7 and GPF-2 chickpea varieties for two consecutive rabi seasons (2015 and 2016). Dual-inoculant treatments; recommended RB-1 + MR33 (4.1%) and RB-1 + MR2 (3.8%) significantly increased the grain yield over Mesorhizobium alone treatments viz MR33 and MR2, respectively. Grain yield in PBG7 variety was significantly affected (7.3%) by the microbial inoculant treatments over GPF2 variety. Therefore, the potential pesticide tolerant strains MR2 and MR33 can be further explored as compatible dual-inoculants with recommended RB-1 for chickpea under environmentally stressed conditions (pesticide application) at multiple locations. Our approach using robust multifunctional pesticide tolerant Mesorhizobium for bio-augmentation of chickpea might be helpful in the formulation of effective bio-inoculants consortia in establishing successful chickpea–Mesorhizobium symbiosis.


Introduction
Chickpea is the most important rabi pulse crop of India with an increase in production from 6.5 million tonnes to 10.3 million tonnes and grain yield from 630 to 935 kg ha −1 during the past 35 years . Inoculation of chickpea seeds with a selected strain of Mesorhizobium sp. is under wide practice to improve the yield by enhancing root nodulation and N uptake of the plant. This helps in reducing the dependency on chemical fertilizers (Brigido et al. 2017). Co-inoculation of plant growth-promoting (PGP) bacteria with Rhizobium synergistically improves the legume growth, symbiotic nitrogen fixation (SNF), yield, and overall plant's performance by absorption of nitrogen, phosphorus and other macro-and micronutrients (Gopalakrishnan et al. 2015). Therefore, identification and manipulation of Communicated by Yusuf Akhter.

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117 Page 2 of 25 their relationships with PGP bacteria might fulfill modern demands of agricultural, economic, social, and environmental sustainability (Hou et al. 2018;Park et al. 2017;Zaid et al. 2020). Chickpea seeds are susceptible to varied fungal pathogens (Ascochyta blight and Botrytis grey mold), insect, termites and weeds. Under a recommended strategy of maximizing productivity, farmers often treat the seeds with fungicides (carbendazim) followed by insecticides (chlorpyrifos) to protect them from seed-borne pathogens and then inoculate them with a nitrogen (N 2 ) fixing strain to use the natural resources. Also, there are two recommended herbicides viz. pre-emergence (pendimethalin) and pre-plant (ready-mix which is a combination of pendimethalin + imazethapyr) herbicides for controlling broad-leaved weeds in chickpea (Anonymous 2013). However, the problems arise when we use chemical pesticide treatments in conjunction with Mesorhizobium/Mesorhizobium + rhizobacterium as biofertilizer. Adverse effects of pesticides on legume-rhizobia symbiosis include inhibition of cell division and nodule formation, limiting carbohydrate supply to the nodules, blocking the initial attachment of complementary rhizobia to lectins by protecting recognition sites present on root hairs and inhibition of symbiotic signaling between host plant and rhizobia (Meena et al. 2020). One of the major constraints faced in Rhizobium inoculant development technology is the selection of isolates that are symbiotically efficient and adapted to the local environmental conditions loaded with various types of stress (Porter et al. 2019). Previous studies have largely focussed on the effects of few pesticides in a limited number of mesorhizobia on SNF in chickpea (Ahemad and Khan 2009Khan , 2011aRathjen et al. 2020). Simultaneously, several studies have been conducted on pesticide tolerant PGP bacteria (Khan et al. 2020;Inthama et al. 2021;Li et al. 2020). However, there is scarce information on pesticide tolerant mesorhizobia in chickpea. The present investigation was designed to screen indigenous Mesorhizobium strains for intrinsic tolerance to recommended pesticides followed by verification of their in-vitro multifunctional traits under pesticide stress. Further, the robust strains of Mesorhizobium with multifunctional traits were explored for ecological performance on plant growth, SNF and yield in chickpea crop raised with the recommended application of pesticides under field conditions.

Procurement and purity of reference cultures
Seventy-five strains of Mesorhizobium sp. including recommended cultures of MR33 (Mesorhizobium cicer sp. LGR33) and PGP rhizobacterium strain RB-1 (Pseudomonas argenttinensis JX239745.1) were procured from the culture collection of Pulses Research Laboratory, Department of Plant Breeding and Genetics, PAU, Ludhiana. The purity of cultures was tested using the Gram staining technique (Ouma et al. 2016). For the differentiation of Mesorhizobium from Agrobacterium and other contaminants, different confirmatory tests were performed on the Hoffer's alkaline (Ansari and Rao 2014), the ketolactose, (Finer et al. 2016), the congo red yeast extract mannitol agar (CRYEMA) and the glucose peptone agar media (Gaur and Sen 1981). The cultures of mesorhizobia and rhizobacterium were maintained on the Yeast Extract Mannitol Agar (YEMA) and the Nutrient Agar (NA) media slants, respectively, and stored at 4 °C for subsequent use during the study period.

In-vitro screening of strains of Mesorhizobium sp. for pesticide tolerance
In this experiment, four recommended pesticides viz. preemergence (pendimethalin) and pre-plant (ready-mix which is a combination of pendimethalin + imazethapyr), insecticide (chlorpyrifos) and fungicide (carbendazim) were tested with selected mesorhizobia strains along with rhizobacterium RB-1 for tolerance to single (1X) and double (2X) the recommended doses of pesticides (Table 1) using the disc diffusion method. Sterile filter paper discs were soaked in pesticide solutions in methanol (based on their optimal solubility) at concentrations equivalent to 1X and 2X recommended doses when applied under field conditions (Table 1). Mesorhizobial and rhizobacterium RB-1 cultures were grown in YEM and nutrient broths, respectively, until they reached the mid-exponential growth phase (OD 600 = 0.8) before inoculation. Pesticide discs were placed on the minimal salt agar medium plates (Ahemad and Khan 2009) which were uniformly spread with the selective bacterial cultures. The plates were kept overnight at 4 °C to allow diffusion of the pesticides, followed by incubation at 28 °C for 3-5 days. Growth of individual bacterial cultures with different pesticides were compared with control discs dipped in sterile water without pesticides. Based on disc diffusion method the bacterial strains (Mesorhizobium strains along with RB-1) that showed luxuriant growth with different pesticides were further tested for their viable count by pour plate method. For this, suspensions of these bacterial strains were prepared in minimal salt broth media supplemented with 1X and 2X doses of the recommended pesticides (Table 1) and incubated at 28 °C for 5-7 days. Serial dilutions of mesorhizobial cultures and RB-1 were plated on YEM and Nutrient agar media, respectively, and incubated at 28 °C for 3-4 days. The bacterial colonies were observed at every fort night. Viable population count of bacteria was calculated as cfu ml −1 by the following formula.
where a = mean number of bacterial colonies, b n = dilution factor, cfu = colony-forming units.
Subsequently, the selected bacterial cultures were grown in different pesticides supplemented with minimal salt broth media for upto 6 months, using sub-culturing technique in 10 ml tubes at 28 ± 2 °C for 48 h and 130 rpm in a rotatory shaker. Mixed culture of each bacterial strain from four different pesticide amended media were taken in LB medium broth incubated for 48 h at 28 ± 2 °C and was termed as the mother culture. This technique was conducted to induce possible mutations in bacterial cultures for intrinsic tolerance to pesticides while maintaining stable multifunctional traits. Further experiments were conducted with the mother cultures of the robust pesticide tolerant strains thus developed in-vitro to assess their consistent performance for the multifunctional traits.

Effect of different pesticides on multifunctional traits in strains of Mesorhizobium sp. (in-vitro)
Indole acetic acid (IAA) production of robust pesticide tolerant strains of Mesorhizobium sp. developed in-vitro was determined by the method as demonstrated by Gordon and Weber (1951) later modified by Brick et al. (1991). For this, Luria Bertani (LB) medium broth cultures of the selected pesticide tolerant strains of Mesorhizobium sp., each having a fixed concentration of tryptophan (100 µgml −1 ) were supplemented with recommended doses of different pesticides in 10 ml tubes (Table 1). It was followed by standard estimation of IAA using orthophosphoric acid and 4 ml of Salkowsky's reagent (2% 0.5 M FeCl 3 in 35% perchloric acid). Selected pesticide tolerant strains of Mesorhizobium sp. were further tested for siderophore production using the Chrome Azurol S (CAS) agar medium supplemented with recommended doses of different pesticides following the method of Schwyn and Neilands (1987). For hydrogen cyanide (HCN) production, all the pesticide tolerant strains of Mesorhizobium sp. were grown on the HCN induction medium (Bakker and Schipper 1987) amended with the recommended dose of individual pesticides. Cell wall degrading enzyme viz. cellulase and protease production by pesticide tolerant strains of Mesorhizobium sp. was analyzed by the formation of clear zones on the carboxymethylcellulose (CMC) agar and skimmed milk (SM) agar medium, respectively, (Vijayabharathi et al. 2018) supplemented with recommended doses of pesticides.

Seed germination and vigor index (in-vitro)
Before the field experiment, compatibility of potential pesticide tolerant strains of Mesorhizobium sp. with rhizobacterium strain RB-1 was tested using the disc plate technique. For this, the sterile filter paper discs were dipped in different mesorhizobial cultures and placed on a lawn of RB-1 rhizobacterial culture, spread on the NA medium. The zone of inhibition around the individual disc indicates the incompatibility of two cultures. This was followed by a seed germination experiment arranged in factorial randomized block design (FRBD) with three replications. Chickpea seeds were treated with the potential strains of Mesorhizobium sp. (MR2, MR17, and recommended MR33) alone and in 6 g a.i. Kg −1 seed *6 9 × 10 3 dual inoculation with recommended RB-1 along with uninoculated control (with pesticides application as per recommendation, alone) as per treatment. For seed treatment, healthy seeds of two chickpea varieties viz. PBG7 and GPF2 were surface-sterilized according to Vincent (1970). Surface sterilized seeds were initially treated with fungicide carbendazim and insecticide chlorpyrifos followed by microbial inoculants as per recommended practice for seed treatment in chickpea; whereas pendimethalin and ready-mix were incorporated in water agar medium simulating their field application (Table 1). Treated seeds were allowed to germinate in Petri dish containing 0.7% water agar with incubation at 28 °C. After 15 days, percent seed germination, fresh weight of seedling, root and shoot length were recorded. Seedling vigor indices were determined by the following formula: Seed vigor index I (SVI) = (mean of the root length + shoot length) × % germination.

Treatment structure for field experiment
The field investigation was conducted for two consecutive winter seasons (2014-15 and 2015-16) in factorial randomized block design (FRBD) with three replications at Pulse Research Farm and Microbiology Laboratory, Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana. The field was prepared with 42 plots having 4 rows each, with row to row distance of 30 cm, plant to plant distance of 10 cm and total plot size of 4 × 2.4 = 9.6 sq.m. Chickpea seeds of desi PBG1 and GPF2 varieties were procured from the Pulses Section, Department of Plant Breeding and Genetics, PAU, Ludhiana. The microbial inoculant treatments comprised of mono-inoculants (potential strains of Mesorhizobium sp. MR2, MR17 and recommended MR33 alone) and dual-inoculants with recommended RB-1 (RB-1 + MR2, RB-1 + MR17 and recommended RB-1 + MR33). The control plants remained un-inoculated with recommended pesticides application alone. For the preparation of monoinoculants, 25 ml of microbial culture was mixed in autoclaved (120 °C, 30 min) charcoal powder base (50 g) and applied to the chickpea seeds at the rate of 20 g charcoal inoculant per kg of chickpea seeds as per treatment. And for the dual-inoculants, a cocktail of Mesorhizobium sp. strain and rhizobacterium strain RB-1 (freshly grown cultures in the YEM and the nutrient media broth, respectively, with a cell density of 10 8 -10 9 CFU ml) was made in the ratio of 1:1 before mixing into charcoal base for seed treatment. Before sowing, inoculated seeds were air-dried at room temperature under the shade and sown within two hours. All the standard agronomic practices were followed for raising the chickpea crop. According to the recommended management practices, chickpea seeds were initially treated with the carbendazim and chlorpyrifos followed by microbial inoculants; whereas ready mix and pendimethalin were applied as pre-plant and pre-emergence herbicides, respectively, in the plots to control the broad-leaved weeds (Table 1).

Plant growth parameters
Emergence count was recorded at days after sowing (10 DAS) from central rows of each plot after leaving two border rows on each side and calculated from the number of emerged seedlings per meter row length. Observations for plant height, dry weight of roots and shoots, chlorophyll (Witham et al. 1971) and carotenoid (Dere et al. 1998) contents were taken after 60 and 90 DAS. For measuring plant height, three plants were randomly selected from each plot and uprooted. Roots were removed from shoots and the height of shoots was measured from the base in cm. Similarly for dry weight of shoots, three randomly selected plants were uprooted from each plot. Roots were detached from shoots, sun dried and then oven dried at 60 °C for 2 days. Dry weight of shoot and root was recorded in g.

Symbiotic nitrogen fixation
Observations of nodule number, dry weight of nodules and leghaemoglobin content were recorded at 60 and 90 DAS. For evaluation of symbiotic parameters, three randomly selected plants were uprooted from each plot with their root system being intact. The roots were washed in running tap water and nodules carefully detached with forceps. The number of nodules per plant was recorded by taking average. The detached nodules were oven dried at 60 °C for 2 days and the dry weight of nodules per plant was recorded in mg. Leghaemoglobin content was estimated according to Wilson and Reisenauer (1963) by reading the absorbance of clear nodular tissue extract using Drabkin's solution at 540 nm.

Antioxidant enzymes activities in root exudates
For the collection of root exudates, frozen root tissues of each treatment were homogenized at 4 °C in an ice-chilled mortar in QB buffer per gram of tissue (Kumar et al. 2009). Crude homogenates were centrifuged at 15,000×g for 15 min at 4 °C, and the supernatant fractions were frozen at − 20 °C. Superoxide dismutase (SOD) activity and catalase (CAT) activity were recorded at 120 DAS by the methods of Aebi (1983) and Marklund and Marklund (1974), respectively.

Soil health
Soil enzyme activities were analyzed at 90 DAS. Estimation of dehydrogenase activity of soil was based on the production of triphenyl formazan (TPF) from triphenyl tetrazolium chloride (Klosse and Tabatabai 2000). Soil Urease activity was calculated as μg of ammonium N released g −1 of soil 2 h −1 (Tabatabai 1982).

Yield attributing traits, seed protein content and grain yield
Yield attributing traits, seed protein content and grain yield were determined at harvesting stage. The number of pods per plant was recorded by taking the average of three randomly selected plants; and the number of seeds per pod was recorded by taking the average of seeds from three randomly selected plants. Crop was sickle harvested and grains were thrashed. Hundred-seed weight was measured on a per-plot basis by taking a sample of the harvested seed in each plot. Total protein content of seeds was determined by Kjeldahl's technique with slight modification (McKenzie and Wallace 1954).

Statistical analysis
Data were analyzed using analysis of variance (ANOVA) based on the pooled mean of two years with SAS Statistical Package Version 9.3. Further, mean separation of treatment effects was accomplished using the Tukey test. The least significant difference (LSD) was calculated at the 5% probability level.

Tolerance of strains of Mesorhizobium sp. with recommended pesticides (in-vitro)
Confirmatory tests based on Hoffer's alkaline medium and ketolactose tests presented white translucent colonies in 50 strains of Rhizobium whereas red colonies indicated Agrobacterium as a contaminant. All the Mesorhizobium strains (50) showed growth at 28 °C on YEMA medium after 48-72 h and were considered as Mesorhizobium. These 50 strains of Mesorhizobium sp. were further subjected to screening for pesticide tolerance along with rhizobacterium RB-1 by the disc diffusion method. The growth inhibition zones around pesticide discs allowed us to divide the bacterial strains under study into three categories, on a three-point scale viz. Luxuriant (+ + +), moderate (+ +) and poor tolerant ( +), owing to their degree of tolerance towards different pesticide treatments. Among the recommended (1X) doses of different pesticide treatments, the highest percentage of luxuriantly tolerant strains were observed with pendimethalin (37%) followed by ready-mix (36%), chlorpyrifos (31%) and carbendazim (30%) (Plate 1). However, only 4% of the strains were poorly tolerant with pendimethallin followed by chlorpyrifos (7%), ready-mix (8%) and 12% with carbendazim. An overall decline in the percentage of luxuriantly tolerant strains was noticed with 2X recommended doses of all the pesticides viz. 2X pendimethalin and 2X ready-mix (by 8% each), 2X chlorpyrifos (by 5%) and 2X carbendazim (by 10%). Comparatively, there was rise in the percentage of poorly tolerant strains with 2X doses of all the pesticides viz. 2X pendimethalin and 2X ready-mix (14% each), 2X chlorpyrifos (20%) and 2X carbendazim (19%). Along with the strains of Mesorhizobium sp., RB-1 also displayed almost luxuriant tolerance towards the 1X doses of all the pesticides while moderate tolerance was shown towards 2X chlorpyrifos and 2X carbendazim. Based on this experiment, 20 strains of Mesorhizobium sp. were found to have the potential of being selected as pesticide tolerant strains. Population density of microbes in the given system is an index of their viability. Viable population count of 20 strains of Mesorhizobium sp. was monitored by exposing them to 1X and 2X doses of recommended pesticides and the data were transformed as log cfu ml −1 (Fig. 1). Among all the bacterial treatments with different pesticides, the highest viable population count was observed for Mesorhizobium sp. strain MR2 with 1X pendimethalin (8.90 log cfu ml −1 ) followed by 1X ready mix (8.70 log cfu ml −1 ), 1X chlorpyrifos (8.63 log cfu ml −1 ) and least with 1X carbendazim (8.53 log cfu ml −1 ). Similar tendency was recorded with the 2X doses of pesticides, however, there was a reduction in the population count of all the bacteria under study. In accordance with this trend, RB-1 also recorded the highest population count with 1X pendimethallin (8.36 log cfu ml −1 ) and least with 2X chlorpyrifos. Conclusively, these tests for intrinsic tolerance of Mesorhizobium strains elucidated the effect of different classes of pesticides on their survival, where the highest inhibitory effect on the growth of Mesorhizobium strains was exhibited by insecticide (chlorpyrifos) followed by fungicide (carbendazim) and herbicides (ready-mix of pendimethalin + imazthpyr and pendimethalin).

sp.
The selected robust strains of Mesorhizobium sp. with intrinsic tolerance to different pesticides developed over several generations in-vitro were further analyzed for multifunctional traits viz. IAA, siderophore and HCN production with recommended pesticide treatments. For IAA production, the selected strains of Mesorhizobium sp., differed significantly with all the pesticides in presence (Trp +) and absence (Trp-) of tryptophan ( Fig. 2a-d).
Additionally, IAA production significantly increased with Trp + over Trp-in different treatments. Among selected strains of Mesorhizobium sp. treated with different pesticides, significantly high IAA production was observed for Mesorhizobium sp. strain MR2 (48.5 µgml −1 ) with pendimethalin (Plate 2) followed by ready-mix, carbendazim and chlorpyrifos (39.8, 25.2 and 23.2 µg ml −1 , respectively). However, an overall decline in IAA production by 1.2 fold was recorded with ready-mix followed by carbendazim (1.6 fold) and chlorpyrifos (2.3 fold) over the control. Siderophore production by the Mesorhizobium strains with recommended pesticides was exhibited by the appearance of clear zones around the bacterial colonies due to the chelation of iron bound to CAS dye (Plate 3a-c). Among 20 pesticide tolerant strains of Mesorhizobium sp., only the strains MR2, MR17 and recommended MR33 were tested positive for siderophore production on CAS medium amended with recommended doses of pendimethalin and ready-mix treatments, whereas, with chlorpyrifos only strain MR2 produced siderophores. However, carbendazim amended CAS agar medium was inhibitory for siderophore production by Mesorhizobium sp. strains MR2, MR17 and recommended strain MR33. The remaining strains of Mesorhizobium sp. appeared negative for siderophore production with all the pesticides under investigation. Further for cell wall degrading enzyme production, among all the pesticide tolerant strains of Mesorhizobium sp., only MR17 was positive for protease production in presence of pendimethalin and ready-mix supplemented SM agar medium while it presented cellulase production with only pendimethalin amended CMC agar medium (Plate 4a-c). However, there was an absence of any cell wall degrading enzyme production in the remaining strains of Mesorhizobium sp. Based on the in-vitro evaluation of multifunctional traits, it was inferred that insecticide (chlorpyrifos) exhibited a highest inhibitory effect on the metabolism of Mesorhizobium strains followed by fungicide (carbendazim) while both the herbicides (pendimethalin and readymix of pendimethalin + imazthpyr) were less toxic towards the selected Mesorhizobium strains, accounting for a comparatively better exhibition of multifunctional traits in the presence of herbicides. These in-vitro results were coincident with the data on survival of the strains with different pesticides validating the consistent multifunctional performance of the robust strains under pesticide stress.

Seed germination and vigor index (in-vitro)
Based on multifunctional traits, two strains of Mesorhizobium sp. viz. MR2, MR17 along with recommended strain MR33 were selected as potential pesticide tolerant strains. In-vitro compatibility test of individual potential pesticide IAA producƟon (μg ml -1 ) Strains of Mesorhizobium sp.

Fig. 2 (continued)
Dual inoculation of MR2 with RB1 further increased the seed germination (1.1 fold), radical length (1.9 fold), plumule length (1.6 fold), fresh weight of seedling (1.4 fold), vigor index I (1.8 fold) and vigor index II (1.5 fold) over un-inoculated control treatment (Tables 2 and 3). However, PBG7 and GPF2 chickpea varieties were at par for seedling germination. All the potential Mesorhizobium strains under study significantly improved the vigor index (1.2-1.3 fold) and germination (2.2-3.4%) which further increased by 1.7-1.8 fold and by 3.4-6.7%, respectively, with individual dual-inoculant treatments over un-inoculated control. However, RB-1 + MR2 performed at par with recommended RB-1 + MR33 for enhancement of seed germination and fresh weight of seedling and vigor index II, whereas, for an increase in plumule length RB-1 + MR17 was at par with recommended RB-1 + MR33. This experiment further confirmed the potential of the selected pesticide tolerant strain of Mesorhizobium sp. viz. MR2 and MR17 along with recommended strain MR33 for enhancement of growth and yield in chickpea. However, the ecological performance of all the selected Mesorhizobium strains alone and individual dual-inoculant treatments with RB-1 was assessed under field conditions for a better understanding of chickpea-Mesorhizobium symbiosis under pesticide stressed field conditions.

Plant growth promotion
Seedling's emergence count has been expressed as percentage (%) germination of seeds. This revealed a significant variation among all the microbial inoculant treatments in respect of the un-inoculated control. With the mono-inoculant treatments, seed germination significantly enhanced, ranged from 5.6 to 8.7% over the un-inoculated control. The highest germination was recorded with recommended Mesorhizobium cicer strain MR33 (92%) followed by Mesorhizobium sp. strain MR2 (91%) and Mesorhizobium sp. strain MR17 (89%) as compared to the un-inoculated control (84%). The germination was significantly enhanced with dual-inoculant treatment of recommended RB-1 + MR33 (4.3%) followed by RB-1 + MR2 (3.3%) over MR33 and  (Table 4). Further, the effect of dual-inoculant treatments on plant height further increased by 25.0-33.9% over the control. In similarity with emergence count, dual-inoculant treatments of pesticide tolerant strains of Mesorhizobium sp. with rhizobacterium RB-1 significantly improved the plant height over Mesorhizobium alone treatments. The highest improvement was obtained with recommended RB-1 + MR33 (13.6%) over MR33 alone followed by RB-1 + MR2 (7.8%) over MR2 alone treatment. Further, PBG7 variety performed significantly better by 10.1% over GPF2 chickpea variety for enhancement of the plant height. A similar trend was followed at 90 DAS, where there was significant variation in the plant height from 8.9 to 17.1% with the mono-inoculant treatments over uninoculated control. All the dual-inoculant treatments, further increased the plant height ranged from 16.5 to 22.7% over un-inoculated control, however, RB-1 + MR2 was at par with RB-1 + MR17. Recommended RB-1 + MR33 presented the highest increase in plant height (11.0%) over MR33 alone treatment. Moreover, plant height significantly increased with PBG7 variety over GPF2 variety by 10.5%. The interaction between variety and treatment was not significant at 60 and 90 DAS, respectively.
The dry weight of shoot (60 DAS), revealed significant variation among all the mono-inoculant treatments over uninoculated control apart from the strain MR17 (Table 4), however, recommended MR33 (14.9%) was at par with MR2 (14.0%). Synchronously, dual-inoculant treatment of RB-1 + MR2 performed at par with recommended RB-1 + MR33 for enhancement of dry weight of shoot. However, the highest increase was significantly obtained with RB-1 + MR2 (18.0%) over MR2 alone followed by recommended RB-1 + MR33 (15.4%) over MR33 alone treatment. Although the effect of treatments on varieties was not significant, yet both the chickpea varieties were at par for the enhancement of dry weight of shoot. Consistent trend was observed at 90 DAS, where the highest dry weight of shoot was significantly recorded with strain MR2 (18.4%) followed by recommended strain MR33 (16.5%) over un-inoculated control. Dual-inoculant treatments RB-1 + MR2 and recommended RB-1 + MR33 led to significantly enhanced dry weight of shoot by 34.1% and 31.6%, respectively, over un-inoculated control. Further, RB-1 + MR2 (13.2%) and recommended RB-1 + MR33 (12.9%) significantly increased the dry weight of shoot over MR2 and MR33 alone treatments, respectively. Both chickpea varieties viz PBG7 and GPF2 were at par for the dry weight of shoot at 90 DAS.
In terms of the dry weight of root at 60 DAS, significant differences were not observed among all the treatments. However, at 90 DAS, all the mono-inoculant treatments resulted in significantly improved root dry weight over uninoculated control apart from Mesorhizobium sp. strain MR17 (Table 4). The increase was witnessed with strain MR2 (8.7%) followed by recommended MR33 (7.6%) over un-inoculated control. All the dual-inoculant treatments For improvement of the chlorophyll content, all the treatments were at par with each other at 60 DAS (Fig. 3). Further at 90 DAS, among all the treatments, significant enhancement in chlorophyll content was seen with RB-1 + MR2 (26.6%) and recommended RB-1 + MR33 (23.8%) over un-inoculated control. However, RB-1 + MR2 and recommended RB-1 + MR33 were at par with each other and also with MR2 and MR33 alone treatments, respectively. The pooled mean analysis also revealed a significant interaction between the varieties and the treatments. However, chickpea variety PBG7 significantly increased the chlorophyll content by 13.7% over GPF2 variety. For carotenoid content of leaves at 60 and 90 DAS, all treatments were at par, and the effect of treatments on varieties was non-significant (Fig. 3). All the dual-inoculant treatments increased carotenoid content by 1.1 fold over the mono-inoculant treatments.
Leghaemoglobin content was also significantly affected by all the treatments except with Mesorhizobium sp. MR17 alone over un-inoculated control (Table 4). However, strain MR2 (16.7%) was at par with the recommended strain MR33 (12.6%) for increasing the leghaemoglobin content.

Antioxidant enzyme activities
All the treatments significantly increased the SOD and CAT activities over the un-inoculated control ( Table 5). The highest increase in SOD activity was recorded with Mesorhizobium sp. strain MR2 (1.3 fold) and the lowest with Mesorhizobium sp. strain MR17 (1.2 fold) over un-inoculated control. However, SOD activity further enhanced by 1.5 fold with dual-inoculant treatment RB-1 + MR2 followed by a 1.4 fold increase with recommended RB-1 + MR33 over uninoculated control. In congruence with SOD activity, significant enhancement in CAT activity was recorded with dualinoculant treatments viz RB-1 + MR2 and recommended RB-1 + MR33 (1.08 fold each) over respective Mesorhizobium alone treatments viz MR2 and MR33. PBG7 variety demonstrated significantly high SOD and CAT activities (1.2 and 1.1 fold, respectively) over GPF2. These results showed that antioxidant enzyme activities were affected more significantly by the Mesorhizobium sp. strain MR2 as compared to recommended MR33 in mono-and dual-inoculant treatments. This presented strain MR2 as a potential candidate for improvement of plant growth and productivity in chickpea under field conditions. It can be explored as an alternative to the recommended MR33 strain in the formulation of biofertilizers in chickpea.

Soil health
In terms of dehydrogenase activity (Table 5), all the treatments differed significantly with un-inoculated control except MR17. However, the recommended strain MR33 (16.1%) and strain MR2 (15.7%) performed at par for improving soil health. The highest effect of dual-inoculant treatments on soil dehydrogenase activity was found with RB-1 + MR2 (33.1%) followed by recommended RB-1 + MR33 (29.7%) while the least was recorded with RB-1 + MR17 (14.2%). Further, recommended RB-1 + MR33 (11.7%) and RB-1 + MR2 (15.0%) significantly increased the soil dehydrogenase activity over MR33 and MR2 alone treatments, respectively. Variety PBG7 performed significantly better by 16.3% over variety GPF2. On the contrary, the strain MR2 performed at par with the recommended strain MR33 for enhancement in soil urease activity at 90 DAS (Table 5). Also, the highest soil urease activity was witnessed with dual-inoculant treatment of strain MR2 with RB-1 (448 μg) followed by recommended RB-1 + MR33 (440 μg) over un-inoculated control (361 μg) while the least was displayed with RB-1 + MR17 (426 μg). Further, RB-1 + MR2 and recommended RB-1 + MR33 significantly increased the urease activity by 7.4% and 5.3% over MR2 and MR33 alone treatments, respectively. PBG7 variety manifested significantly high urease activity (2.5%) over GPF2 variety. This also indicated that the strain MR2 positively affected the soil enzyme activities closely followed by recommended strain MR33 with improvement on dual inoculation with RB-1.

Total protein content of seeds, yield attributing traits and grain yield
In terms of the number of pods, significant differences existed among all the treatments except MR17 compared to the un-inoculated control ( Table 6). All the dual-inoculant treatments were at par with each other, however, an enhanced The number of seeds per pod also revealed significant differences for all the treatments, however, the interaction between variety and treatment was non-significant (Table 6). In mono-inoculant treatments, the highest improvement in the number of pods was observed with strain MR2 (16.5%) while strain MR17 (6.7%) displayed the least increase over un-inoculated control. Further, among dual-inoculant treatments, enhancement in the number of pods was highest with RB-1 + MR2 (29.3%) followed by recommended RB-1 + MR33 (23.2%) and RB-1 + MR17 (20.1%) over uninoculated control. The number of seeds further enhanced by 10.9% with RB-1 + MR2 over MR2 alone followed by 8.0% with recommended RB-1 + MR33 over MR33 alone. Means of varieties (PBG7 and GPF2) were at par for the number of pods.
All the treatments showed significant variation for the hundred seed weight except MR17 alone treatment (Table 6). However, strain MR2 (12.0%) and the recommended strain MR33 (6.8%) were at par with each other for the improvement of seed weight as compared to un-inoculated control. The highest seed weight was observed with RB-1 + MR2 (15.0%) followed by recommended RB-1 + MR33 (9.8%) and RB-1 + MR17 (9.8%) over un-inoculated control. However, all the dual-inoculant treatments were at par with individual Mesorhizobium alone treatments for improvement of the seed weight. There was a significant effect of PBG7 variety on the enhancement of seed weight by 11.8% over GPF2 variety. Protein content of seeds did not show any significant difference with the microbial treatments and the varieties. Among mono-inoculant treatments, the highest protein content was observed with MR33 (20.2%) followed by MR2 (20.0%) and MR17 (19.7%) as compared to uninoculated control (18.9%). While, among dual-inoculant treatments, the maximum protein content was recorded with RB-1 + MR2 (21.4%) followed by recommended RB-1 + MR33 (21.1%) and RB-1 + MR17 (20.5%). An additional increase in protein content of seed was noticed with dual-inoculants viz. RB-1 + MR2 and recommended RB-1 + MR33 by 1.04 fold each over MR2 and MR33 alone treatments, respectively. For grain yield, a significant difference was observed with all the mono-inoculant treatments except MR17 (Table 6) while recommended MR33 (7.2%) was at par with MR2 (6.5%) over un-inoculated control. However, in dual-inoculant treatments, significantly high grain yield was registered with recommended RB-1 + MR33 (11.5%) followed by RB-1 + MR2 (10.6%) and least was presented with RB-1 + MR17 (6.5%) over un-inoculated control. In corroboration with growth and symbiotic parameters, dual-inoculant treatments viz. recommended RB-1 + MR33 (4.1%) and RB-1 + MR2 (3.8%) significantly increased the grain yield over Mesorhizobium alone treatments viz MR33 and MR2, respectively. Grain yield in PBG7 variety was significantly affected (7.3%) by the microbial inoculant treatments over GPF2 variety.

Discussion
Development of biofertilizer technology faces limitation in the selection of isolates that are symbiotically efficient as well as adapted to the local environmental conditions. Alternatively, much of the interest has been diverted towards the exploration of bacteria with the ability to exploit xenobiotic compounds as growth substrates (Ataikiru et al. 2020;Bhatt et al. 2020;Birolli et al. 2019). Our study was based on the approach of induction of tolerance in rhizobial inoculants towards the recommended pesticides in legume host, while sustaining consitent PGP traits for improving plant growth and productivity under ecologically compromised conditions. Effect of pesticides on legume-rhizobia symbiosis depends on rhizobial species, type, and the concentration of pesticide (Kumar et al. 2010). Results from in-vitro screening experiment also revealed appreciable differences among strains of Mesorhizobium sp. for tolerance to different pesticides and doses; where significantly high tolerance was observed with 1X doses of herbicides viz. pendimethalin and ready-mix followed by fungicide (1X carbendazim) and insecticide (1X chlorpyrifos). Higher tolerance with pre-emergence herbicide (pendimethalin) was possibly obtained through the decomposition of complex N-containing pendimethalin molecule as a proteinaceous source providing both C and N to the microbes (Cycon and Pitrowska-seget 2016). A similar mechanism might have contributed to the tolerance in bacterial inoculants with pre-plant herbicide ready-mix, which is a combination of pendimethalin + imazethapyr. As per the observations of Drouin et al. (2010), herbicide imazethapyr was not found to have any inhibitory effect on all the Rhizobium strains under study. Survival of all the bacterial strains was significantly affected with different pesticides treatment over the untreated samples. However, in alignment with our study, a significant reduction in the number of rhizobia was recorded with fungicides (captan and thiram) by Dunfield et al. (2000). Also in another study, a significant reduction in rhizobia was observed in combined seed treatment of chickpea with fungicide (captan) and insecticides (endosulfan and chlorpyrifos) (Kunal and Sharma 2012). Similarly, in our experiment, the highest inhibitory effect towards the growth of strains of Mesorhizobium sp. was presented by the insecticide (chlorpyrifos) as compared to other pesticides. Further, an overall decline in the viable population count of bacteria with double doses of different pesticides might be due to their toxic effect mainly on the moderately and poorly tolerant strains. Thus, the survival of different bacterial strains to different pesticides can be correlated to the function of pesticide degradation encoded in their genome combined with the evolutionary process (Briceno et al. 2020;Hawkins et al 2019;Pileggi et al 2020).
The rhizosphere is a zone of intense molecular interactions due to deposition of the root exudates, that supports higher microbial growth than the surrounding soil known as the "rhizosphere effect" (Badri et al. 2009). The influence of bacteria in the rhizosphere is largely due to the production of auxin phytohormones (indolic compounds) which depends on L-tryptophan as the main precursor (Moreira et al. 2016). Etesami et al. (2015) indicated the importance of IAA in nodulation events as it helps to circumvent the plant defence system for successful host colonization. Therefore, IAA production becomes one of the important parameters for assessing the effectiveness of rhizobia in plant growth promotion and nodulation. It has been reported by Raut et al. (2017) that IAA production varies from bacteria to bacteria and pesticide to pesticide. Further, Kulandaivel and Nagarajan (2014) confirmed that the strains of Pseudomonas and Azospirillum produced IAA for up to 1.25% concentration of pesticide endosulfan while Klebsiella strain inhibited the IAA production. Similar variations in the IAA production by Mesorhizobium strains with different pesticides in our study was explained on the basis of inhibition or change in IAA producing metabolic pathways by the pesticides. Moreover, utilization of tryptophan as the precursor for IAA biosynthesis accounted for the significant increase in the IAA production with tryptophan supplementation in all the treatments. Further, on an overall basis, among different pesticides, a significant increase in the IAA production with pendimethalin treatment as compared to the control was ascribed to the utilization of pendimethalin as a substrate for the production of the energy required for the synthesis of IAA. However, a significant reduction in the IAA production with the readymix, followed by chlorpyrifos and carbendazim as compared to the control could be due to the imposition of stress or toxic effect of these pesticides on the biosynthetic machinery of Mesorhizobium strains involved in the production of important biomolecules like IAA. Such a variation of IAA in Mesorhizobium strains with different pesticides is well supported by the study of Drouin et al. (2010), who noticed different effects of various classes of pesticides (insecticides, herbicides and fungicides) on rhizobia.
Iron (Fe) is an essential micronutrient for plants and microorganisms, which is involved in various important biological processes, such as photosynthesis, respiration, chlorophyll biosynthesis and SNF. Bacteria can overcome the nutritional Fe limitation by using chelating agents called siderophores that form a complex with Fe 3+ which may be absorbed by plant species for partial Fe nutrition. Competition for iron acquisition among rhizobia determines successful persistence in the rhizosphere leading to efficient host colonization (Raines et al. 2016). In our study on siderophore production under pesticide stress, the pesticide tolerant Mesorhizobium sp. strain MR2 was observed to be more promising followed by recommended strain MR33 and strain MR17. These results are in confirmation with the work of Ahemad and Khan (2012) who reported siderophores-production by Mesorhizobium sp. strain MRC4 on CAS agar medium amended with insecticides (fipronil and pyriproxyfen). Cell wall degrading enzymes like the protease and cellulase are produced by rhizobia which play a role in the degradation of the plant cell wall for infection thread formation and also in antagonism against pathogenic fungi. These results were supported by our in-vitro antagonistic effect against Fusarium wilt (unpublished work). In congruence with our findings, protease production has been reported in rhizobia strains by Oliveira et al. (2010). Also, Robledo et al. (2012) purified a cell-bound bacterial cellulase (CelC 2 ) enzyme from Rhizobium leguminosarum bv. trifolii nodulating the clover. In contrast to these studies, we observed production of the cell wall degrading enzyme by the rhizobial strains under study also in the presence of pesticides. Further, the compatibility of individual pesticide tolerant strains of Mesorhizobium sp. with the recommended culture of PGP strain rhizobacterium RB-1 along with a recommended application of pesticides was analyzed through in-vitro seed germination assay. As suggested by Shakir et al. (2016), the presence of pesticides in soil may affect the uptake of essential nutrients by plant root. Functional groups like -OH, -NH2, -CO.NH2, -COOR and -NR-, in pesticides can accelerate the process of adsorption in the soil which may affect the growth and development of plant via disrupting the soil water plant relationship (Shakir et al. 2016). This causes nutrients deficiency thereby retarding the plant growth by inhibiting various physiological processes like seed germination, seedling growth, cell division, cell elongation and enlargement, and tissue and organ differentiation retorted growth (Ju et al. 2020). Moreover, several studies have demonstrated a significant enhancement in seed germination and seedling vigour in chickpea-Rhizobium on co-inoculation with PGP bacteria (Abdel Fattah 2015;Kaur et al. 2015;Verma et al. 2013) on account of increased bioavailability of nutrients such as P and K along with stress mitigation and release of plant growth hormones with PGP bacteria. As per our findings, also, significant improvement in germination and vigor index was noticed with potential pesticide tolerant strains of Mesorhizobium sp. alone as compared to the un-inoculated control which further significantly improved with individual dual inoculation treatments. Since the combination of microbial inoculants and different pesticides may have synergistic or antagonistic interplay (Murturi et al. 2017). In accordance with our in-vitro results, the herbicides (pendimethallin and ready-mix) might have acted in synergism with the pesticide tolerant Mesorhizobium strains, that further synchronized with the dual-inoculant treatments. This counteracted the harmful effect of other pesticides (carbendazim and chlorpyrifos) combined pesticide application. Similar ecological interactions decided the plants' response under field conditions. Plant height indicates the growth index of the crop. Production of PGP substances like IAA affects many physiological activities of plants including cell enlargement, cell division, root initiation, growth rate, phototropism, geotropisms, and apical dominance and also the symbiotic Rhizobium-legume interaction. Liu et al. (2017) reported that effective IAA producing isolates can stimulate plant growth due to their early colonizing ability to out-manoeuvre the plant defence mechanisms. This is well in line with the present study, where inoculation with efficient IAA producing strains of Mesorhizobium sp., MR2,MR17 and MR33 resulted in increased plant height, dry weight of shoot and root over un-inoculated control. Already, it has been established that the PGP bacteria belonging to various genera such as Bacillus, Pseudomonas help in plant growth promotion in direct and indirect ways such as improving the soil properties and biological activities (Ahmad et al. 2016;Etesami 2018). In addition to this, several reports confirm that synergism between PGP bacteria and Rhizobium promote plant growth and development, supporting our results for synergistic improvement in the plant growth parameters in dual-inoculant treatments as compared to single inoculation (Hussain et al 2019;Ju et al 2020). As demonstrated by Giménez-Moolhuyzen et al. (2020), pesticides produce negative effects on crop physiology-especially on photosynthesis-leading to a potential decrease in both the growth and the yield of crops. Conversely, the combination of PGP bacteria and Rhizobium have already been used by several workers for their potential in alleviating damage to plant tissues and improve soil biological activities under xenobiotic stress (Antoniadis et al. 2017;Ju et al. 2019;Sipahutar et al. 2018). Evidently, in our study significant enhancement in plant growth was noticed with all the treatments despite the combined application of pesticides as compared to the uninoculated control (with recommended pesticides application alone). This was correlated to the production of IAA by the strains of Mesorhizobium sp. under pesticide stress in our in-vitro experiment. Our results are in corroboration with Ahemad and Khan (2012) who attributed the improvement in plant growth parameters and productivity in chickpea to IAA production in pesticide tolerant Mesorhizobium sp. strain MRC4. In line with these findings, our results for the increased chlorophyll content with dual-inoculant treatments of Mesorhizobium strains with RB-1 as compared to their mono-inoculations are related to efficient uptake of nutrients such as Mg (an important part of chlorophyll molecule) and N and Fe (play role in photosynthesis and enzymes involved in chlorophyll synthesis) mediated by the PGP bacteria. It is in close confirmation with Kannan et al. (2015) who reported a positive interaction between root colonization, nutrient uptake and growth promotion of plants.
Nodulation is an important trait for effective SNF in leguminous plants, however, pesticides have been reported to trigger root hair deformations and inhibit symbiosis (Fox et al. 2007). In the present investigation, efficient nodulation caused by pesticide tolerant strains of Mesorhizobium sp. might be caused by counteraction of phytotoxic effects of pesticides by their biodegradation or enzymatic hydrolysis (Martani et al. 2011). Our results are in agreement with Ahemad and Khan (2011b) for improvement of nodulation parameters (nodule number and nodule dry weight) with herbicide-tolerant PGP strain of Bradyrhizobium MRM6 over the un-inoculated plants in greengram. Moreover, in a previous study, Gonzalez et al. (1996) showed that herbicide imazethpyr did not have any direct influence on Rhizobium spp. and decline in pea nodulation was attributed to the effect on plant growth rather than on rhizobia. Similarly in our study, the intrinsic tolerance of strains of Mesorhizobium sp. towards pesticides assisted in the amelioration of the harmful effects of pesticides. In line with this, Etesami et al. (2015) established that IAA production by bacteria in root nodules activates H + -ATPase required for energy production in nodules. Further, Ghosh et al. (2015) correlated the production of IAA and other PGP substances by Rhizobium undicola to the plant-microbe interaction and nodule function in Neptunia oleracea. These findings are in close corroboration to our results on improved nodule biomass and N fixation with multifunctional pesticide tolerant strains of Mesorhizobium sp. as compared to the uninoculated control. Concomitantly, substantial production of PGP substances by rhizobacterium RB-1 further improved plant growth and development by creating more infection sites. This accounted for the significant increase in nodulation with dual inoculation treatments in relation to the monoinoculations. Synthesis of O 2 scavenging leghaemoglobin is an index of efficient SNF in legumes as it maintains high levels of ATP (Diez-Mendez et al. 2015). As per our results, higher nodulation and N fixation with dual-inoculant treatments might have increased the nodule occupancy leading to increased leghaemoglobin content over mono-inoculant treatments. Moreover, the combined effect of multifunctional traits of potential pesticide tolerant Mesorhizobium strains and PGP strain RB-1 viz. siderophores mediated uptake of iron which is a constituent of leghaemoglobin protein, might have accorded to the increased leghaemoglobin content in dual-inoculant treatments as compare to the mono-inoculation treatments (Gonzalez et al. 2016). Our results are well supported by Korir et al. (2017) where co-inoculation of Rhizobium and Paenibacillus polymyxa enhanced leghaemoglobin concentration, nitrogenase activity, and N fixation efficiency in common bean. Batista et al. (2016) postulated that facilitated nutrient uptake by PGP bacteria along with phosphate solubilization (that provides P for ATP) and siderophores mediated uptake of iron (that acts as e − carrier in nitrogen fixation reaction carried out by nitrogenase enzyme), enhanced the fixed N that was incorporated as proteins. Similarly, in our study, an increase in protein content with dual-inoculant treatments might be attributed to the increased availability of fixed N that was further explained by facilitated uptake of micronutrients such as Mo acting as a cofactor in nitrogenase enzyme. This is further correlated to IAA production, which helps in nutrient uptake with the proliferation of roots (Haris and Ahmad 2017). Similar reports on co-inoculation with Rhizobium and PGP bacteria have shown improvement in seed protein and N content in soybean (Hungria et al. 2015;Masciarelli 2014) and chickpea (Verma et al. 2013).
Legumes and other N 2 -fixing plants face oxidative risks beyond those associated with photosynthesis. As is the case with leaves, nodules are rich in strong reducing compounds, polyunsaturated fatty acids and O 2 -labile proteins (e.g. nitrogenase) that can readily react with O 2 and generate reactive oxygen species (ROS). Application of agrochemicals like pesticides induce a significant amount of abiotic stress resulting in the production of ROS that leads to the disruption of cellular activities. Antioxidant enzymes mainly superoxide dismutase enzyme (SOD) and catalase (CAT) play a major role in protecting the cell from oxidative damage by scavenging the ROS and optimizing cell function by regulating cellular redox state and modifying gene expression (Sidhu et al. 2018;Singh et al. 2021). Superoxide dismutase enzyme converts superoxide anion-free radical (O 2 ) to H 2 O 2 . On the contrary, catalase enzyme plays a role in converting H 2 O 2 to oxygen and water. Our results clearly indicated that mono-inoculated chickpea with pesticide tolerant strains of Mesorhizobium sp. presented significantly high SOD and CAT activity against ROS formed in response to the environmental stresses generated under field conditions. This confirmed their robustness for environmentally stressed conditions correlating with their intrinsic tolerance towards pesticides. There are several reports on induction of antioxidant stress response in the host plants by the PGP bacteria (Anzuaya et al. 2017;Francisco et al. 2020;Hasanuzzaman et al. 2020). Alternately, in-vitro experiments revealed highest production of exopolysaccharides with the strain MR2 (869 µgml −1 ) as compared to other strains in presence of pesticides (2X ready mix) (unpublished work). Therefore, it could provide possible explanation for an additional increase in the antioxidant enzyme activities in dual-inoculated over the mono-inoculated plants with RB-1 + MR2 treatment, as a protective mechanism against pesticide-stress under field conditions. Therefore, these results demonstrated the significance of the synergistic effect of dual inoculations in mitigating the abiotic stress delivered by pesticides in the host plant by increasing the activities of SOD and CAT in scavenging ROS. Our observations are in congruence with Fatnassi et al. (2015) who reported enhanced SOD activity with combined inoculation of Rhizobium + PGP bacteria as compared to the control in Vicia faba. Similarly, an increase in SOD and CAT activities has been reported in the co-inoculation of Piriformospora indica, Pseudomonas sp. and Mesorhizobium cicer over single inoculation with P.indica and Mesorhizobium alone in chickpea (Mansotra et al. 2015). In addition to this, Dong et al. (2014) observed that in petroleum contaminated soils, the co-inoculation of PGP bacterium Serratia marcescens + AMF Glomus intraradices significantly improved the SOD activity over uninoculated control. However, an overall decrease in catalase as compared to SOD activity in our study was attributed to more consumption of the catalase enzyme to detoxify ROS (Park et al. 2017). In a more recent study, an overall induction of antioxidant protective mechanisms in alfalfa associated with Cd stress tolerance was observed in response to PGP rhizobacterium Pseudomonas fluorescence and P. indica co-inoculation (Sepehri and Khatabi 2021).
Soil enzymes viz urease and dehydrogenase are important indicators of soil microbial activity and play a role in nutrient (N and H) recycling. Several reports have established that pesticides designed to kill organisms can adversely affect the soil health and quality by altering the microbial community structure (Brtnicky et al. 2019;Danso Marfo et al. 2019;Ren et al. 2019). However, the overall effect of combination of pesticides on microbes is subjected to interaction between pesticides (synergistic, additive or antagonistic). Additionally, host-symbiont-related modifications in the rhizosphere shifts the microbial community composition and size, as well as enzyme dynamics (Blonska et al. 2016). Our findings for the significant increase in soil enzyme activities with pesticide tolerant strains of Mesorhizobium sp. is well supported by the work of Siczek and Lipiec (2016) where high soil urease and dehydrogenase activity in faba bean with Rhizobium over control was attributed to changes in microbial community structure and enzymatic processes in response to rhizobia. Corresponding to other growth and SNF parameters in our study, a significant increase in soil enzyme activities with all dual-inoculant treatments were observed. Synergistic interactions among dual-inoculants and the chickpea plant helped in the development of a higher root system which led to the accumulation of root exudates in the rhizosphere attracting higher microbial populations for mediating the soil enzyme activities. Since pesticides damage non-target microorganisms via directed interference with host metabolism and via also oxidative stress mechanisms. Further, a mixture of different pesticides reduce the microbial diversity and alter the community structure as explained by Murturi et al. (2017). In the present study collectively, the positive stress response system induced in chickpea by the combination of potential pesticide tolerant Mesorhizobium strains and rhizobacterium strains assisted in achieving the desired effect for enhancing the soil health and plant productivity under pesticide stress.
Results on plant growth and SNF, are well in line with our study on the significant enhancement in the yield attributes and grain yield. This could be due to effective symbiosis with potential pesticide tolerant and multifunctional strains of Mesorhziobium. Our findings are supported by Ahemad and Khan (2009) where a significant improvement in yield components following fipronil-and pyriproxyfen-tolerant Mesorhizobium inoculation are reported in chickpea. In a similar study, significant improvement in plant growth and productivity was accomplished with pesticide tolerant Rhizobium phaseoli strain in chickpea (Rathjen et al. 2020). Additional significant improvement in yield attributes with dual-inoculant treatments was attributed to an increase in the number of branches with PGP rhizobacterium RB-1 with a concomitant increase in yield attributing traits such as the number of pods, seeds and ultimately the grain yield. In addition to this, dual-inoculant treatments improved photosynthetic rates due to improvement in nutrient and water absorption due to IAA mediated augmented root system. This led to higher nutrient assimilation in grain with increased seed weight and grain yield. This is in congruence with several studies reporting significant enhancement in chickpea productivity and yield on co-inoculation with various PGPR strains under ecologically stressed conditions (Benjelloun et al. 2021;Verma et al. 2010;Vurukonda et al. 2016). Overall, in different chickpea genotypes, improvement in growth parameters, yield attributing traits and grain yield might be the result of differential interaction of variety with microbial inoculants which could be further attributed to variation in rhizospheric community structure in response to root exudates (Nasr Esfahania et al. 2016).

Conclusions
Current agricultural practices in Punjab have witnessed indiscriminate use of pesticides (including herbicides, insecticides and fungicides) due to which ecological performance of bio-inoculants may be compromised resulting in a reduction in the yield and productivity of the legumes. In the present study, in-vitro developed bacterial strains for pesticide tolerance revealed three robust strains of Mesorhizobium sp. viz. MR2 and MR17 and recommended MR33 that showed the highest plant growth promotional activities. Although, under laboratory conditions, strains MR2 and MR17 performed significantly better over the recommended strain MR33 for PGP traits, yet under field investigation, Mesorhizobium sp. strain MR2 was at par with recommended strain MR33 for enhancement of plant growth, SNF and yield in chickpea over un-inoculated control. Further dual-inoculant treatments with recommended PGP strain RB-1 significantly enhanced all the parameters. The potential pesticide tolerant strains MR2 and MR33 can be further explored as compatible dual-inoculants with recommended RB-1 for chickpea under environmentally stressed conditions (pesticide application) at multiple locations. Therefore, screening for robust multifunctional pesticide tolerant Mesorhizobium might open new avenues for improving chickpea productivity in pesticide-stressed soils of Punjab. Overall, these findings postulate that bio-augmentation of chickpea with multifunctional Mesorhizobium with improved pesticide tolerance might be helpful in the formulation of effective bio-inoculants consortia in establishing successful chickpea-Mesorhizobium symbiosis. However, concerted research and development are needed to integrate engineered microorganisms in the agro-ecosystem for sustainable agriculture.