Mesorhizobium Ciceri as a Biological Tool for Improving Physiological, Biochemical and Antioxidant State of Cicer Aritienum L. by Lowering the Fungicide Induced Oxidative Stress


 The present study demonstrates the interactions of fungicide-tolerant symbiotic bacteria Mesorhizobium ciceri with Cicer arietinum-kitazin (KITZ) in greenhouse conditions. Under both in vitro and soil systems, KITZ imparted deleterious impacts on plants as a function of dose. The three-time KITZ dose detrimentally and maximally reduced germination efficiency, vigor index, dry matter production, symbiosis, leaf pigments and seed attributes of C. arietinum. KITZ- induced morphological alterations in root tips, oxidative damage and cell death in root cells of C. arietinum were shown by SEM. M. ciceri tolerated up to 2400 µgmL− 1 of KITZ, synthesized considerable amounts of bioactive molecules including indole-3-acetic-acid (IAA), 1-aminocyclopropane 1-carboxylate (ACC) deaminase, siderophores, exopolysaccharides (EPS), HCN and ammonia, and solubilised inorganic phosphate even in fungicide-stressed media. Following application to soil, M. ciceri improved performance of C. arietinum and enhanced dry biomass production, yield, symbiosis and leaf pigments even in a fungicide-polluted environment. At 92 µgKITZkg− 1 soil, M. ciceri maximally and significantly (p ≤ 0.05) augmented whole plant length by 41%, total dry biomass by 18%, carotenoid content by 9%, LHb content by 21%, root N by 9%, shoot P by 11% and pod yield by 15%. Additionally, M. ciceri was associated with decreased levels of stressor molecules (proline and MDA) and antioxidant defence enzymes (APX, GPX, CAT and POD) of C. arietinum plants when inoculated in soil. The symbiotic strain effectively colonized the plant rhizosphere/rhizoplane. In pesticide- contaminated soils, inoculation of M. ciceri may serve as an excellent strategy for augmenting C. arietinum productivity.


Introduction
Cicer arietinum L. (chickpea) crops often suffer from attack by phytopathogens, which damage the crop and consequently limits crop yield. Fungicides are commonly used to enhance productivity by preventing phytopathogen-related damage . However, massive and injudicious use of such chemicals can upset soil fertility and inhibit microbial communities (Walia et al., 2014) and enzymatic activities (Han et al., 2020). Apart from the cytotoxic and genotoxic effect of fungicides on soil microbiota, uptake and translocation of pesticides by different plant organs may severely damage important metabolic activities leading subsequently to death of plants (Eker et al., 2006). Exceptionally high concentrations of pesticides disrupt: (i) cellular organelles and membrane permeability (Shahzad et al., 2018); (ii) respiratory processes and carbohydrate metabolism (Kumar et al., 2012); (iii) physiologically active enzymes (Liu et al., 2006) and proteins (Yin et al., 2016); (iv) photosystems by blocking the effective quantum yield of PSII (ΦPSII) and quantum e ciency of PSII (Fv/Fm) (Niinemets and Kull, 2001); and (v) cause oxidative damage (Singh and Roy, 2017) and genetic makeup of cellular machinery (Gill and Tuteja, 2010). Zablotowicz and Reddy (2007) observed that the pesticide glyphosate considerably decreased nitrogenase activity of rhizobia. As a consequence, symbiotic events leading to nodule formation and root morphogenesis of plants were drastically diminished (Adami et al., 2017). In another study, the fungicide pyrimorph was found to strongly inhibit the electron transport (ET) reactions of chloroplasts and adversely affected the physiology of whole plants (Xiao et al., 2014).
To overcome these problems, certain physico-chemical approaches have been used to destroy pesticides in soil. However, these methods are expensive, and the remediation process often remains incomplete due to the transformation of the parent compound to metabolites which are sometimes more persistent and more toxic for non-target organisms than were the parent compounds. As a consequence, physicochemical methods of pesticide removal have not been widely accepted. Alternative methods of pesticide degradation/detoxi cation are therefore necessary. Bioremediation offers some solutions to pesticide detoxi cation problems. This technique, often referred to as 'microbial remediation' relies on the identi cation of microorganisms to convert contaminants to simpler and harmless forms and hence, to mitigate pesticide pollution.
To this end, scientists have identi ed pesticide degrading/detoxifying microbes endowed with potential plant growth promoting activities. Chief among them belongs to genera Ensifer (Ma et  Given the nutritive importance of C. arietinum in the diet, the negative impact of fungicides on crop productivity, the lack of adequate information on fungicidal response to C. arietinum and the bioremediation potential of PGPR, this study was formulated. The objectives were to: (i) assess the fungicidal toxicity to C. arietinum both under in vitro bioassays and pot-house conditions; (ii) assess the kitazin-induced distortion, oxidative damage and cell death in C. arietinum root cells; (iii) isolate and identify the symbiotic bacterium from C. arietinum root nodules; (iv) evaluate fungicidal tolerance by nodule bacteria; (v) determine the production of bioactive molecules under fungicide stress; (vi) evaluate the effects of M. ciceri on physiological and biochemical attributes of C. arietinum; (vii) determine the stressor molecules and antioxidant enzymes from C. arietinum foliage detached from fungicide-treated and bio-inoculated plants; and (viii) evaluate the rhizosphere/rhizoplane colonization potential of M. ciceri. LV, JEOL, Japan) following the treatment of roots on soft agar (0.7% w/v) amended with 1000 µg mL − 1 KITZ. After seven days of germination, roots were picked up from the agar plates and rinsed at least thrice with sterile phosphate buffer saline (PBS) and then xed and processed following our previously described protocol for tissue xation for SEM (Ahmed et al., 2018). Fixed and ethanol dehydrated tissues were examined under SEM at 10 kV accelerating voltage to check the fungicide induced distortion/damage in root tips, if any. A control set was also included for comparison. procedure was followed (Ref). C. arietinum roots grown in the presence of three concentrations (1X, 2X, and 3X) of KITZ were allowed to take up the 0.25% w/v solution of Evans blue stain for at least 15 min. After gentle washing with DDW thrice, emission of uorescence was examined under LSM-780.

Isolation of Root Nodule Bacteria and Kitazin Tolerance
Fresh and un-damaged healthy nodules were detached from Cicer arietinum (chickpea) plants and surface sterilized by dipping nodules in 4% NaOCl for 2 min., washed three times with sterile double distilled water (DDW) and crushed gently. A-100 µL freshly extracted nodule suspensions were streaked on YEMA plates and incubated at 28 ± 2 0 C for 3-5 days. A total of 20 Mesorhizobium strains were isolated and morphologically and biochemically identi ed (Holt et al., 1994). Plant infection technique was carried out to determine the host speci city (Vincent, 1970). Mesorhizobial isolates were exposed to varying concentrations of kitazin using minimal salt agar (MSA) in order to select pesticide tolerant Mesorhizobium strain. Colonies grown on YEMA plates and e ciently surviving at the highest concentration of pesticides were chosen and referred to as pesticide tolerant mesorhizobial strains (PTMS). Of the total 20 Mesorhizobium isolates, BRM5 expressing maximum tolerance to pesticides was selected.

Molecular Identi cation of BRM5 Isolate
For the identi cation of isolate to genus level, 16S rRNA sequencing was performed  The IAA produced by Mesorhizobium ciceri BRM5 was quantitatively assessed by modi ed method of Brick et al. (1991). For the assay, strain M. ciceri was cultured in LB broth containing a xed amount (100 µg mL − 1 ) of tryptophan and amended with 600 (1X), 1200 (2X) and 1800 (3X) µg mL − 1 of KITZ (See supplementary methods for details).
The isolate was spot inoculated on kitazin supplemented universal chrome azurol S (CAS) agar plates followed by incubation at 28 ± 2 °C for detection of orange colour halo around the bacterial colonies. Also, the siderophore was quantitatively assessed by growing the bacterial strain in fungicide amended iron (Fe) free succinate liquid medium as suggested by Barbhaiya and Rao (1985). The estimation of siderophore was done according to universal chrome azurol liquid assay (Schwyn and Neilands 1987). Siderophore units were calculated as follows: For ACC deaminase activity, M. ciceri was cultured in broth supplemented with various kitazin concentrations and amount of α-ketobutyrate produced by strain was determined following the method of Honma and Shimomura (1978) and Penrose and Glick (2003) (See supplementary methods).

EPS production, HCN and Ammonia Production
Exopolpysaccharide (EPS) produced by M. ciceri under fungicide stress was scrutinized by culturing the cells into liquid medium supplemented with variable doses of KITZ (see supplementary method for details). The production of cyanogenic compound (HCN) and ammonia were assayed using the methods of Bakker and Shipper (1987) and Dye (1962), respectively.

Planting, Fungicide Treatment and Application of Mesorhizobium
Seeds were disinfected/sterilized with NaOCl (2%), washed, cleaned and desiccated at room temperature.
Commercial grade fungicide kitazin (Table S1) [recommended dose: 1 × (96 µgKg − 1 ), 2 × (192 µgKg − 1 ) and 3 × (288 µgKg − 1 )] of soil were applied to moist experimental soils before sowing of seeds (at least one day before sowing). The soils were lled in 20 × 24 cm clay pots of having approximately 5 kg soil per pot. Seeds were then coated/bacterized with freshly prepared inoculum of M. ciceri after dipping the seeds in liquid culture medium for 2 h using 10% gum arabic as a sticker to achieve 1 × 10 8 cells seed − 1 which was con rmed by viable cell count. The un-inoculated sterilized seeds submerged in sterile water only were taken as control. Non-bacterized and bio primed seeds (n = 10) were sown in respective earthen pots containing 5 kg of conventional soils. Two controls were run in parallel; one was un-inoculated and untreated control (without bacteria and without fungicides) and another was inoculated (only bacteria but no fungicides). Each test concentration was replicated thrice and pots were arranged in a completely randomized block design. After germination, seedlings were thinned and two uniform healthy seedlings of C. arietinum were maintained in each pot, 15 days after emergence (DAE). Pots were watered regularly and were kept in open eld condition. The crop experiments were carried out regularly for two succeeding years to achieve the consistency in results.
2.6.2 Germination E ciency, Plant height, Dry biomass and Photosynthetic Pigment in the presence of M. ciceri and KITZ The KITZ treated and bacterized C. arietinum plants were removed at 80 and 120 DAS and germination e ciency, root and shoot length, weight and dry biomass was measured. For dry biomass, plants were dried in oven (Yorco, York Scienti c Industries, Pvt. Ltd. India) at 80 0 C for 2 days and then weighed using an electronic scale balance (BL-220 H, Shimadzu, Japan), and average was calculated. Leaf photosynthetic molecules (chlorophyll and carotenoid) accumulated in fungicide treated/untreated and bacterized C. arietinum foliage was estimated following the methods of Arnon (1949) and Kirk and Allen (1965), respectively (See supplementary methods in electronic supporting information).

Symbiosis, Nutrient Uptake and Seed attributes
Symbiotic features of C. arietinum were assayed by carefully removing the nodules from root systems. Nodules were counted and oven dried (80 °C) in a ventilated oven for 48 h. After drying, nodule dry biomass (mg plant − 1 ) was weighed using an electronic scale balance (BL-220 H, Shimadzu, Japan) and average was calculated. Furthermore, leghaemoglobin (LHb) content was quantitatively assayed following the earlier demonstrated method of Shahid and Khan (2018). (see electronic supporting information).
The nutritional content (nitrogen and phosphorous) in fungicide treated and bacterized C. arietinum plants was estimated at harvest as previously described by Jackson (1976) and Lindner (1944), respectively. Seed yield was recorded. Grain protein was extracted and estimated following the method of Lowry (Lowry et al., 1951). (See supplementary methods).   (1982), respectively. All enzyme assays were performed three times with three replicates of each assay.

Rhizosphere and Rhizoplane Colonization by M. ciceri under Stress
The colonization of root surface by M. ciceri was determined in the presence/absence of fungicide. For the examination, roots were rinsed with DDW and PBS. The scanning electron microscopy was performed following the method of . Furthermore, the colonization of roots in term of CFU g − 1 of root material was determined at 40 and 80 DAS after exposure with different concentrations of fungicides.

Statistical analyses
The data were statistically analyzed using Sigma Plot 12.0 and Minitab17 software. Complete randomized block design (CBRD) for pot experiments was followed with at least three pots per individual test concentration. Crop experiments were conducted for two consecutive years to con rm the reproducibility of data. The data recorded in each year were pooled and analyzed. The mean of the data within a single column was calculated and compared with control treatments. The data represented either in gures or tables is the mean ± standard deviation (S.D.) of at least three replicates (n = 3). Different alphabets in graphs and tables show a signi cant difference among the treatments at a con dence level of p ≤ 0.05. The least signi cant difference (LSD) among treatment means was calculated by two-way analysis of variance (ANOVA) at p ≤ 0.05.

Germination Percentage, Vigor Index and Plant Length
The impact of dose of KITZ on germination e ciency and seedling attributes of C. arietinum seed developed on fungicide-amended agar plates was variable but negative ( Fig. 1) The 3X concentration showed pronounced toxicity and signi cantly (p < 0.05) reduced the germination %, vigor index (SVI), and radicle (RL) and plumule (PL) length by 40%, 47%, 66% and 79% compared to control, respectively (Figs. 1 panel a-c). The reduction in germination e ciency and vigor index may possibly due to the distressed germination metabolism caused by the fungicide. Pesticides detrimentally in uenced the germination ability of different legumes as reported by various workers. In this regard, lethal effect of fungicides on seedling germination and biological attributes of P. sativum under in vitro conditions has been reported ).
3.1.2 Phytotoxicity %, Tolerance index (TI) and Root-Shoot Length Ratio A 19%, 42% and 88% phytotoxicity was recorded for C. arietinum when grown with 96, 192 and 288 µg KITZ kg − 1 , respectively, compared to control (Fig. 1e). The higher KITZ concentration (3X) maximally affected the root-shoot length ratio and reduced it by 0.9 − 0.4 (55% reduction over control) (Fig. 1d). The tolerance index (TI) in C. arietinum decreased with increasing KITZ doses and con rmed a negative correlation between fungicide and TI. The TI of C. arietinum was recorded at 70, 56 and 26% at 1X, 2X and 3X dose of KITZ respectively; over the untreated control ( Fig. 1 panel f). These results indicate that lower fungicide concentrations resulted in maximum TI, whereas the 3X dose exhibited the minimum TI in C. arietinum. Similarly, root-shoot length ratio and tolerance index of chickpea were negatively in uenced by the higher concentrations of two neonicitnoid group of pesticides (Shahid et al., 2020). Fungicide-induced oxidative stress in root membranes was also visualized. AO/PI stained and fungicidetreated C. arietinum roots were observed using confocal laser scanning microscopy (CLSM). A concentration-dependent increase in dead/injured cells observed as red/orange color occurred in roots exposed to 3X of KITZ ( Fig. 2 panel III B1 B2 and B3). Untreated root tissues exhibited maximum intensity of green uorescence resulting from AO reaction representing little or no damage (Fig. 2 panel III B). This is an indication that pesticide exposure was arbitrated by ROS-mediated damage to membrane lipids which therefore increased uorescence of DNA-bound propidium iodide in membranes. Cortés-Eslava et al. (2018), using CLSM, reported similar oxidative stress, oxidative damage and apoptosis in two model plants grown in insecticide-stressed conditions. The loss/damage of plasma membrane in fungicidetreated root tissue was obvious when C. arietinum roots were stained with Evans blue dye. The uptake of dye by root tissues increased three-to four-fold with increasing KITZ concentrations ( Fig. 2 panel IV C1, C2 and C3). In contrast, dye was not taken up by untreated roots (Fig. 2 panel IV C) and hence, the root margin remained smooth signifying its functional integrity.

Biochemical and Molecular Identi cation of Mesorhizobium and Fungicide Tolerance
Strain M. ciceri was characterized morphologically and biochemically (Table S2). Based on biochemical and cultural characteristics, the genus of the symbiotic bacterium was con rmed and strain was presumed as Mesorhizobium. Isolate BRM5 showed the maximum base sequence similarity (> 96.7%) to type strain Mesorhizobium jarvisii ATCC 233669 T (Accession number NR135858.1), (Fig. S1). Based on this relatedness, isolate BRM5 was identi ed as Mesorhizobium ciceri.
The tolerance of M. ciceri to KITZ was assessed while grown in minimal salt (MS) broth added with variable concentrations of fungicide; strain BRM5 survived up to 2400 µgmL -1 of KITZ (Table S2) (Shahid et al., 2019b) have also been reported. Secretion of IAA by the fungicide-tolerant BRM5 strain even at higher levels of pesticide is a promising feature of soil microbes, because such pesticide-tolerant PGPR strains, when used in harsh environments are likely to endure producing/releasing phytohormones such as IAA. This crucial growthaugmenting plant hormone will thus be accessible to plants even at high levels of pesticides. A trend similar to IAA was observed for siderophore production. M. ciceri synthesized siderophores even under stressed conditions (Fig. 3A panel b-d).  (Fig. 3A panel e). Secretion of ACC deaminase by the tolerant strain, however, even in stressed environments is agronomically a bene cial feature for increasing productivity of crops under pesticide stress. This intrinsic property of ACC deaminase production even under pesticide pressure makes them a promising choice for crop production even in soils polluted with excess pesticide.

C. arietinum-Fungicide-Mesorhizobium Interactions: Comprehensive Toxicity and Bioremediation Studies
Bioinoculation impact of M. cicero on Biochemical Characteristics of C. arietinum

Seed Germination
The kitazin tolerant M. ciceri improved the growth of plants when applied to C. arietinum plants in soil system treated with variable level of fungicide ( Fig. 4 panel a). The impact of M. ciceri BRM5 on germination e ciency of C. arietinum seedlings grown in earthen pots supplemented separately with varying doses of KITZ was variable ( Fig. 4 panel b). Generally, strain BRM5 had a positive impact on germination and vigor index relative to un-inoculated seeds. M. ciceri BRM5 exhibited a maximum increase of 5% and 6% in germination and SVI at 96 µgKITZkg − 1 (Fig. 4

Dry Biomass Accumulation
The bacterized and un-inoculated C. arietinum plants cultivated in soil treated with varying levels of fungicide had variable dry biomass of C. arietinum. A gradual increase in root and shoot biomass of M. ciceri BRM5-inoculated plants treated with different doses of KITZ was observed both at 80 and 120 DAS. M. ciceri BRM5 maximally increased root, shoot and total dry biomass by 12, 17 and 18%, (at 80 DAS) when applied in soil treated with 96µgKITZkg − 1 compared to dry biomass of un-inoculated but treated with the same dose of KITZ (Table S3, Fig. 4 panel e). There was a signi cant (p ≤ 0.05) interaction between application of symbiotic bacterium and fungicide. The effect of bio-priming and fungicide on biological and chemical characteristics of test plants was signi cantly correlated both at 80 DAS and 120 DAS as revealed by regression analysis and principal component analysis (PCA) (Fig. S1 and S2).

Bio-inoculation Impact of M. ciceri on Photosynthetic Molecules
The  (Table S3) The roots detached from un-inoculated and KITZ treated C. arietinum showed the poorly developed root system and weak/unhealthy nodular systems. In contrast, a better root system having healthy and more pink-colored showing wavy margin was recorded in bio-inoculated C. arietinum plant (Fig. 5 panel a, b). Generally, the symbiotic attributes [nodule number (NN) and nodule dry biomass (NDB)] of M. ciceri BRM5 bacterized C. arietinum plants grown in the presence of KITZ was greater compared to those recorded for un-inoculated plants supplemented with the identical dose of fungicide. M. ciceri BRM5 maximally increased NN and NDB by 23% and 22% at 80 DAS and 16% and 14%, respectively at 120 DAS when used with 96 µgKITZkg − 1 compared to un-inoculated but KITZ-treated plants (Fig. 5 panel c and d). Ullah et al. (2016) also reported that PGPR strains in combination with M. ciceri increased the growth and nodulation of C. arietinum even in pesticide-stressed conditions.

Leghaemoglobin (LHb) Content and nutrient uptake in nodule
The LHb content in fresh nodules of C. arietinum declined in the presence of symbiotic bacteria similar to that in plants grown in fungicide only-supplemented soils. A considerable and 6% in pod number, pod weight, seed number and seed yield was recorde when M. ciceri BRM5 was used with 96 µgKITZkg − 1 soil over un-inoculated but pesticide-treated control (Fig. 6 panel a and b). Likewise, BRM5 increased the grain protein by 7% when used with the 1X dose of KITZ compared to uninoculated plants treated with the identical dose of fungicide (Fig. 6 panel c). The decrease in protein content of grain is likely be due to the binding of pesticides to R-SH groups of proteins which, in turn, alters protein structure. However, seed features of C. arietinum were generally improved following inoculation with M. ciceri BRM5 even in the presence of pesticide. R. leguminosarum strain PS1, when used as bio-inoculant in pesticide-treated pea plants, increased SY by 43% compared with fungicidetreated but un-inoculated plants (Tariq et al., 2016). Enhancement in growth attributes and yield of atrazine-treated Phaseolus vulgaris when grown in the presence of a consortium containing Rhizobium sp. and Trichoderma have been reported (Madariaga-Navarrete et al., 2017). Similar improvements in growth and nutrient levels were observed when stress-resistant PGPR strains of P. aeruginosa and Burkholderia gladioli were were applied to plants grown under stressed conditions (Khanna et al., 2019).

Nutrient Uptake
Bio-inoculation impact of M. ciceri BRM5 on N and P content of C. arietinum plant organs at 80 DAS differed in a concentration-dependent manner. The N content in C. arietinum roots increased from 20.6 to 22.6 µg g − 1 whereas in shoot tissue it increased from 11.5 to 13.3 µg g − 1 when M. ciceri BRM5 was used in the presence of 96 µgKITZkg − 1 (Fig. 6 panel d). Similarly, M. ciceri BRM5 at 96 µgKTZkg − 1 soil improved root and shoot P by 7 and 11%, respectively, compared to non-inoculated plants treated with similar concentrations of KTZ ( Fig. 6 panel e). Uptake of nutrients (N and P) by C. arietinum raised in pesticide-enriched soils was also enhanced following inoculation with fungicide-tolerant symbiotic bacteria. In a similar study, ACC deaminase positive PGPR strains Pseudomonas brassicacearum Am3, P. marginalis Dp1 and Rhodococcus sp. Fp2 were found to improve growth and uptake of both major and trace nutrients viz., N, P, K, Ca, S and Fe in different varieties of legumes raised in stressed soils (Safronova et al., 2006). In another study, Phaseolus vulgaris plants inoculated with stress-tolerant PGPR belonging to a group of phosphate-solubilizing bacteria signi cantly lowered electrolyte leakage, LPO level, SOD, hydrogen peroxide and proline phosphatase activities and improved physio-biochemical attributes, nutrient uptake, and protein and carbohydrate content by relieving the stress (Rady et al., 2019).

Proline and MDA Content
The BRM5-inoculated C. arietinum plants had low levels of proline, MDA and antioxidant enzyme activity in organs even in the presence of different concentrations of KITZ. In general, strain BRM5 minimized the proline level even in the presence of fungicide. M. ciceri BRM5 signi cantly (p ≤ 0.05) and maximally reduced the proline content in roots, shoots and seeds by 27%, 26% and 33% at the 1X dose of KITZ ( Fig. 7 panel a and b) compared to un-inoculated plants treated with the identical dose of fungicide.
Similarly, it was observed that M. ciceri BRM5 reduced the MDA content from 5.4 to 3.07 µ moles g − 1 fw compared to un-inoculated plants treated with 96 µgKITZkg − 1 (Fig. 7 panel c). Information is lacking as to how and why proline levels decline in legumes bio-inoculated with pesticide-tolerant PGPR and raised Similarly, bacterial strains maximally lowered the POD activity by 21% (from 2.78 to 2.2 µ mol min − 1 mg − 1 fw) in the foliage system when detached from 1X concentration of KITZ ( Fig. 7 panel d). Likewise, the APX and GPX activities of C. arietinum were increased by 19.6% (from 2.09 to 1.68 µ mol min − 1 mg − 1 fw and 9% (from 0.99 to 0.90 µ mol min − 1 mg − 1 fw respectively at the 3X dose of KITZ following application of symbiotic bacterium (Fig. 7 panel e). In a similar study, strain SRB02 of B. aryabhattai considerably reduced levels of oxidative stress and antioxidant enzymes CAT, POD and SOD in soybean plants grown in stressed soil, and promoted overall growth of plants (Park et al., 2017). The declines in antioxidants due to inoculation with pesticide-tolerant bacterial strains consequently resulted in a substantial upsurge in overall growth of C. arietinum even under pesticide stress.

Rhizosphere and Rhizoplane Colonization by Mesorhizobium under Stress
Root colonization is a critical initial component of plant-microbe interaction in the plant rhizosphere. This mutualistic interaction is helpful in growth and development of plants as well as in protecting the crops from various biotic and abiotic insults (Verma et al., 2018). Considering this, halotolerant PGPR strain M. ciceri was checked for its root colonizing ability using SEM in the absence (Fig. 8A) and presence of KITZ (Fig. 8B). SEM images revealed that fungicide-untreated roots resulted in dense/compact colonization whereas treated roots showed lesser bacterial populations. Similar colonization of bacteria on C. arietinum root surfaces and consequent increase in plant growth was reported by other workers (Alekhya and