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Alleviation of Cadmium Adverse Effects by Improving Nutrients Uptake in Bitter Gourd through Cadmium Tolerant Rhizobacteria

Alleviation of Cadmium Adverse Effects by Improving Nutrients Uptake in Bitter Gourd through... environments Article Alleviation of Cadmium Adverse E ects by Improving Nutrients Uptake in Bitter Gourd through Cadmium Tolerant Rhizobacteria 1 1 1 , 2 , 3 , Muhammad Zafar-ul-Hye , Muhammad Naeem , Subhan Danish *, Shah Fahad * , 4 , 5 6 4 , 7 , 8 Rahul Datta * , Mazhar Abbas , Ashfaq Ahmad Rahi , Martin Brtnicky , 8 9 10 Jir ˇí Holátko , Zahid Hassan Tarar and Muhammad Nasir Department of Soil Science, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan 60000, Pakistan; zafarulhyegondal@yahoo.com (M.Z.-u.-H.); ch.naeem276@gmail.com (M.N.) Department of Agronomy, The University of Haripur, Haripur 22620, Pakistan College of Plant Sciences and Technology, Huazhong Agriculture University, Wuhan 430070, China Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, Faculty of AgriSciences, Mendel University in Brno, 61300 Brno, Czech Republic; martin.brtnicky@mendelu.cz Institute of Horticultural Sciences, Faculty of Agriculture, University of Agriculture, Faisalabad 38000, Pakistan; rmazhar@hotmail.com Pesticide Quality Control Laboratory, Multan 60000, Pakistan; rahisenior2005@gmail.com Institute of Chemistry and Technology of Environmental Protection, Brno University of Technology, Faculty of Chemistry, Purkynova 118, 62100 Brno, Czech Republic Department of Geology and Pedology, Faculty of Forestry and Wood Technology, Mendel University in Brno, 61300 Brno, Czech Republic; jiri.holatko@centrum.cz Soil and Water Testing Laboratory, Mandi Bahauddin 50400, Pakistan; zahidtarar123@yahoo.com Soil and Water Testing Laboratory for Research, Multan 60000, Pakistan; swt_mltn@yahoo.com * Correspondence: sd96850@gmail.com (S.D.); shah.fahad@mail.hzau.edu.cn (S.F.); rahulmedcure@gmail.com (R.D.) Received: 16 May 2020; Accepted: 22 July 2020; Published: 26 July 2020 Abstract: Cadmium is acute toxicity inducing heavy metal that significantly decreases the yield of crops. Due to high water solubility, it reaches the plant tissue and disturbs the uptake of macronutrients. Low uptake of nutrients in the presence of cadmium is a well-documented fact due to its antagonistic relationship with those nutrients, i.e., potassium. Furthermore, cadmium stressed plant produced a higher amount of endogenous stress ethylene, which induced negative e ects on yield. However, inoculation of 1-amino cyclopropane-1-carboxylate deaminase (ACCD), producing plant growth promoting rhizobacteria (PGPR), can catabolize this stress ethylene and immobilized heavy metals to mitigate cadmium adverse e ects. We conducted a study to examine the influence of ACCD PGPR on nutrients uptake and yield of bitter gourd under cadmium toxicity. Cadmium tolerant PGPRs, i.e., Stenotrophomonas maltophilia and Agrobacterium fabrum were inoculated solely and in combination with recommended nitrogen, phosphorus, and potassium fertilizers (RNPKF) applied under di erent concentration of soil cadmium (2 and 5 mg kg soil). Results showed that A. fabrum with RNPKF showed significant positive response towards an increase in the number of bitter gourds per plant (34% and 68%), fruit length (19% and 29%), bitter gourd yield (26.5% and 21.1%), N (48% and 56%), and K (72% and 55%) concentration from the control at di erent concentrations of soil cadmium (2 and 5 mg kg soil), respectively. In conclusion, we suggest that A. fabrum with RNPKF can more ecaciously enhance N, K, and yield of bitter gourd under cadmium toxicity. Keywords: ACC deaminase; heavy metal stress; PGPR; fertilizers; nutrients; yield Environments 2020, 7, 54; doi:10.3390/environments7080054 www.mdpi.com/journal/environments Environments 2020, 7, 54 2 of 16 1. Introduction High use of pesticides, inorganic fertilizers, and untreated sewage water has significantly contributed to the buildup of heavy metals in agricultural soils [1,2]. These heavy metals become part of soil at the exchange site and remain readily available for plants. Rapid industrialization and anthropogenic activities are also allied factors responsible for the accumulation of toxic metals beyond their threshold limit in cultivatable lands [3–6]. Among di erent heavy metals, cadmium (Cd) is an acute toxin due to its high resistance time, i.e., >1000 years and water solubility [7]. Presence of cadmium below 0.5 mg kg soil is considered a safe limit, but depending upon parent material, it can 1 1 be accumulated up to 3.0 mg kg soil [8]. Being a part of phosphate fertilizers (up to 4.4 mg kg ), it is easily taken up by crops as Cd-supplement [9,10]. Cadmium causes cardiovascular, respiratory, cancer, and renal, skeletal system in humans when taken up beyond the threshold limit [11,12]. The high concentration of Cd in plant tissues disturbs nutrient uptake and creates water imbalance that results in poor photosynthesis [13]. It also causes lipid membrane instability, alteration in membranes permeability, and chlorosis in plants [14,15]. Due to its divalent nature, it competes with divalent essential nutrients, i.e., P, Ca, Mg, and decreases their uptake in plants [16–18]. Bioavailability of K is also a ected when Cd is present in higher concentration in soil [19]. Heavy metal toxicity and physiochemical properties soil depend on the land use [20–22]. Di erent crops, the biological adsorption factor (mg Cd/kg plant ash, mg Cd/kg soil) based on Cd content in plant ash, is di erent, i.e., winter wheat grains (5.97) and straw (2.50), barley grains (4.06) and straw (2.50), sugar beet roots (4.63) and tops (1.41), pea beans (3.22) and straw (0.88), corn grains (8.75) and straw (2.53), soya beans (4.31) and straw (4.63), sunflower seeds (10.8) and stem (4.28) [23]. Moreover, biosynthesis of endogenous stress ethylene under Cd toxicity plays a notorious role that aids in poor root growth [24–26]. In addition, ethylene (C H ) is a plant-signaling molecule. It is involved in seed germination 2 4 flower senescence, root elongation, fruit ripening, and leaf abscission. Mostly ethylene is synthesized in a two-step process, i.e., (1) enzymatic conversion of S-adenosyl methionine (SAM) to 1-amino cyclopropane-1-carboxylic acid (ACC); (2) conversion of ACC to ethylene, which is catalyzed by ACC-oxidase [27]. However, synthesis of endogenous ethylene level is significantly enhanced upon exposure of plants to abiotic stresses, including low soil fertility [28,29]. This endogenous stress ethylene negatively a ects gas exchange attributes, nutrients and water uptake, and yield of di erent crops under any stress conditions [30,31]. To overcome these problems, inoculation of ACC deaminase producing plant growth promoting rhizobacteria (PGPR), could be an ecacious and nature friendly technique [32–36]. Certain PGPRs can improve growth attributes of crops under heavy metals toxic conditions by secretions of ACC deaminase, siderophores, indole acetic acid, gibberellins, and better availability of water and nutrients [37–40]. Enzyme ACC deaminase cleaves stress ethylene into intermediate compounds; thus, decreases the stress generating factors in plants [41,42]. Among di erent crop plants, bitter gourd is a rich source of vitamins, carbohydrates, and proteins [43,44]. As compared to cucumber and tomato, one cup of bitter gourd juice (94 g) has 93% reference daily intake (RDI) of vitamin C [45]. It is cultivated in Pakistan (6107 hectares), with an annual production of 57,190 tons [46]. However, the yield of bitter gourd is negatively a ected when cultivated in Cd pollution. As improvement in N, P, and K can mitigate the stress of Cd toxicity in plants [3], which is why the current study was conducted to explore the ecacy of ACC deaminase producing PGPR with recommended NPK fertilizers (RNPKF) on bitter gourd nutrients uptake and yield under Cd toxicity. Environments 2020, 7, 54 3 of 16 The present study aimed to explore (1) e ectiveness of rhizobacteria in the improvement of nutrients uptake; (2) e ect of nutrients on bitter gourd yield under cadmium-induced stress; (3) correlation of inorganic fertilizer with rhizobacteria on yield and nutrients attributes of bitter gourd under Cd stress. We hypothesized that ACC deaminase-producing PGPRs could improve nutrient uptake and alleviate adverse e ects of Cd in bitter gourd for yield improvement. 2. Materials and Methods 2.1. Experimental Site and Treatments A pot experiment was conducted in the Department of Soil Science research area, Bahauddin Zakariya University, Multan, Pakistan. The soil was characterized as dark brown and saline with JAKHAR soil series [42]. Six treatments were applied in four replication by following two factorial completely randomized designs (CRDs). The treatments were control (without NPK or bacterial strains), recommended NPK fertilizers (RNPKF), Stenotrophomonas maltophilia, Agrobacterium fabrum, RNPKF + S. maltophilia, and RNPKF + A. fabrum. All treatments were added in the soil at 2 and 5 mg Cd kg soil. Artificial toxicity of Cd was developed by using analytical grade salt of CdCl [25]. As per treatment plan, two levels of Cd were maintained, i.e., 2 and 5 ppm (mg kg soil), keeping in mind the Cd concentration of pre-experimental soil. Rhizobacteria were inoculated at the time of sowing. However, required fertilizers were applied at the time of pot preparation. 2.2. Collection of Bacterial Strains and Broth ACC deaminase producing PGPRs S. maltophilia (ACC deaminase activity = 71.78 mol 1 1 -ketobutyrate mg protein h ) and A. fabrum (ACC deaminase activity = 432.6 mol -ketobutyrate 1 1 mg protein h ) were taken from the Laboratory of Soil Microbiology, Department of Soil Science. Both PGPRs were documented Cd tolerant previously, i.e., survive over 5.0 mg Cd kg soil toxicity [25]. For seeds inoculation, Dworkin and Foster (DF) media without agar was used for inoculum preparation [47]. Loop of each rhizobacteria was taken in the sterilized flask and incubated at 25  3 C and 100 rpm for 48 h. After that, broth optical density (OD) was measured by spectrophotometer (540 nm wavelength). Finally, dilution was made with sterilized distilled water to 7 8 –1 achieve 0.45 nm OD, to achieve a uniform population of 10 –10 colony forming units (CFU) mL . 2.3. Seeds Sterilization and Sowing HgCl (0.1%) solution was used for sterilization of seeds. All seeds were placed for 5 min in the solution followed by, three times, washing with sterilized deionized water [48]. Moreover, 1mL respective PGPR inoculum was used for seeds inoculation along with sugar (30% sucrose), peat, and clay (1:1) in 1:2:6 ratio for 100 g seeds. Four inoculated seeds were sown in each pot. Sowing of bitter gourd seeds was done by hand. After 20 days of sowing, only three healthy seedlings were maintained in each pot by thinning. 2.4. Irrigation and Fertilizer Application In pots, 65% field capacity was maintained on a weight basis during the experiment. To fulfil the requirement of crop nutrients (187N, 75P, and 225K kg ha ) urea, K HPO and K SO were applied. 2 4 2 4 2.5. Harvesting and Samples Analyses Harvesting was done at the time of fruit maturity. Samples were digested for the determination of biochemical attributes. The number of bitter gourds was counted manually. For fruit length, standard measuring tape was used. For determination of yield per plant, fruits were collected and weighed on the analytical balance. With the help of diacid mixture nitric acid and perchloric acid (2:1 ratio), the tissues of the plant were digested for P and K analyses [49]. Phosphorus in the samples was determined by using ammonium molybdate and ammonium metavanadate yellow color method [50]. Environments 2020, 7, 54 4 of 16 However, for analyses of K in samples, the digested solution was run on flame photometer [51]. For determination of nitrogen, samples were digested in concentrated H SO , and digestion mixture 2 4 (K SO (100):CuSO .5H O (10):FeSO (1)). Distillation was performed in Kjeldahl distillation apparatus, 2 4 4 2 4 Environments 2020, 7, x; doi: FOR PEER REVIEW  4 of 16  using boric acid as a collector [52]. 2.6. Statistical Analyses  2.6. Statistical Analyses One‐way  ANOVA  was  used  to  assess  the  effects  of  treatments.  Two  factorial  ANOVA  was  One-way ANOVA was used to assess the e ects of treatments. Two factorial ANOVA was conducted  separately  to  compare  PGPRs  and  RNPK  interaction  under  different  levels  of  Cd.  conducted separately to compare PGPRs and RNPK interaction under di erent levels of Cd. Treatment comparison was computed at p ≤ 0.05 by Tukey’s Test.  Treatment comparison was computed at p  0.05 by Tukey’s Test. 3. Results  3. Results 3.1. Number of Bitter Gourds per Plant 3.1. Number of Bitter Gourds per Plant  E ects of PGPRs and RNPKF under di erent levels of Cd were significant (p  0.05) on the Effects  of PGPRs and  RNPKF under  different levels  of  Cd  were significant  (p ≤ 0.05)  on  the  number of bitter gourds per plants (BDP). Inoculation of PGPRs and RNPKF have significant main and number of bitter gourds per plants (BDP). Inoculation of PGPRs and RNPKF have significant main  −1 interactive and interact e iv ects e effect on BDP s on BDP at 2 and  at 2 5an mg d 5kg  mg kg soil. soil. Application  Application of RNPKF of RNPKF + S. + S. maltophilia  maltophilia , RNPKF , RNPKF +  A. fabrum, RNPKF, S. maltophilia and A. fabrum showed significant positive e ect over control at 2 and + A. fabrum, RNPKF, S. maltophilia and A. fabrum showed significant positive effect over control at 2  1 1 −1 −1 5and mg 5 Cd  mg kg Cd kg soil so foril BDP for BDP (Figur  (Fig eure 1). 1). Interaction  Interaction between  between PGPRs  PGPRs and  and RNPKF  RNPKF at at 2 2 mg  mg Cd  Cdkg kg  soil soil  −1 (Figur (Figure e 2 A) 2A) and  and 5 mg 5 mg Cd Cd kg kgsoil so (Figur il (Figeure 2B) 2B) wer were e significant  significant ordinal  ordinal for BDP  for BD (Figur P (F eig 2B). ureIt 2B) was . It noted  was  that noted Cd tha showed t Cd showed non-significant  non‐signnegative ificant negative correlation  correlation (0.1451;  (−0p.14 =51 0.3986) ; p = 0.with 3986)BDP  with . However  BDP. How , PGPR ever,  (0.5863; PGPR (p0.586 = 0.0002) 3; p = 0.000 and 2) RNPKF  and RN (0.3237; PKF (0p.3= 237 0.0541) ; p = 0.054 showed 1) showe positive d positive significant  significa corr nt elation  correlation with with BDP  BDP (Figure 3). The maximum increase of 34% and 68% in BDP was observed from control where  (Figure 3). The maximum increase of 34% and 68% in BDP was observed from control where RNPKF + 1 −1 A.RN fabrum PKF +was  A. fa applied brum wa ats 2ap and plied 5 mg at 2Cd  and kg 5 mg soil,  Cdr kg espectively  soil, respectively. .   2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil a a ab a a 16 ab ab ab bc Control RNPKF S. A. fabrum RNPKF + S. RNPKF + A. maltophilia maltophilia fabrum Treatments Figure 1. Number of bitter gourds per plant (BDP) treated with plant growth promoting rhizobacteria Figure 1. Number of bitter gourds per plant (BDP) treated with plant growth promoting rhizobacteria  (PGPRs), recommended NPK fertilizers (RNPKF), and their combination under 2 and 5 mg Cd kg −1 (PGPRs), recommended NPK fertilizers (RNPKF), and their combination under 2 and 5 mg Cd kg   soil. Di erent small letters express significant di erences (p  0.05). soil. Different small letters express significant differences (p ≤ 0.05).     No. of Bittergourds / Plant Environments 2020, 7, x; doi: FOR PEER REVIEW  5 of 16  Environments 2020, 7, 54 5 of 16 Environments 2020, 7, x; doi: FOR PEER REVIEW  5 of 16  −1 −1 Estimated Marginal Means of BDP 2 mg Cd kg  soil  Estimated Marginal Means of BDP 5 mg Cd kg  soil  −1 −1 Estimated Marginal Means of BDP 2 mg Cd kg  soil  Estimated Marginal Means of BDP 5 mg Cd kg  soil        (A)  (B)  (A)  (B)  p < 0.05  p < 0.05  p < 0.05  p < 0.05  Figure 2. Interaction graph of S. maltophilia (NS1), A. fabrum (NS2), and RNPKF at 2 (A) and 5 mg Cd  Figure −1  2. Interaction graph of S. maltophilia (NS1), A. fabrum (NS2), and RNPKF at 2 (A) and 5 mg Cd  Figure 2. Interaction graph of S. maltophilia (NS1), A. fabrum (NS2), and RNPKF at 2 (A) and kg  soil (B) for number of bitter gourd per plant (BDP).  −1 1 kg  soil (B) for number of bitter gourd per plant (BDP).  5 mg Cd kg soil (B) for number of bitter gourd per plant (BDP). Number of Bitter Gourd per Plant Number of Bitter Gourd per Plant 0.70 0.70 0.5863* 0.60 0.5863* 0.60 0.50 0.50 0.3986 0.40 0.3986 0.3237* 0.40 0.3237* 0.30 0.30 0.20 0.20 0.10 0.0541 0.0002 0.10 0.0541 0.0002 0.00 0.00 Cd PGPR RNPKF Cd PGPR RNPKF -0.10 -0.10 -0.20 -0.1451ns -0.20 -0.1451ns Figure 3. Pearson correlation of Cd, PGPRs, and RNPKF for number of bitter gourd per plant (BDP). Figure 3. Pearson correlation of Cd, PGPRs, and RNPKF for number of bitter gourd per plant (BDP).  * = significant (p  0.05); ns = non-significant. Figure 3. Pearson correlation of Cd, PGPRs, and RNPKF for number of bitter gourd per plant (BDP).  * = significant (p ≤ 0.05); ns = non‐significant.  * = significant (p ≤ 0.05); ns = non‐significant.  3.2. Bitter Gourd Fruit Length 3.2. Bitter Gourd Fruit Length  3.2. Bitte E ects r Gou ofrPGPRs d Fruit Len inoculation gth  and application of RNPKF under various Cd levels were significant (p  0.05) on bitter gourd fruit length (FL). Application of RNPKF + S. maltophilia and RNPKF + Effects of PGPRs inoculation and application of RNPKF under various Cd levels were significant  Effects of PGPRs inoculation and application of RNPKF under various Cd levels were significant  A. fabrum were significantly di erent from control at 2 and 5 mg Cd kg soil for FL. It was observed (p ≤ 0.05) on bitter gourd fruit length (FL). Application of RNPKF + S. maltophilia and RNPKF + A.  (p ≤ 0.05) on bitter gourd fruit length (FL). Application of RNPKF + S. maltophilia and RNPKF + A.  −1 that RNPKF showed a positive significantly better response at 2 mg Cd kg soil but remained fabrum were significantly different from control at 2 and 5 mg Cd kg  soil for FL. It was observed that  −1 fabrum were significantly different from control at 2 and 5 mg Cd kg  soil for FL. It was observed that  −1 non-significant at 5 mg Cd kg soil over control for FL (Figure 4). Main e ects of PGPRs and RNPKF  showed  a  positive  significantly  better  response  at  2  mg  Cd  kg   soil  but  remained  non‐ −1 RNPKF  showed  a  positive  significantly  better  response  at  2  mg  Cd  kg   soil  but  remained  non‐ −1 RNPKF were significant, but their interaction remained non-significant for FL at 2 and 5 mg kg soil. significant at 5 mg Cd kg  soil over control for FL (Figure 4). Main effects of PGPRs and RNPKF were  −1 significant at 5 mg Cd kg  soil over control for FL (Figure 4). Main effects of PGPRs and RNPKF were  −1 Disordinal non-significant interaction was found between PGPRs and RNPKF at 2 mg Cd kg soil, but significant, but their interaction remained non‐significant for FL at 2 and 5 mg kg  soil. Disordinal  −1 significant, but their interaction remained non‐significant for FL at 2 and 5 mg kg  soil. Disordinal  −1 the interaction was non-significant ordinal at 5 mg Cd kg soil for FL. Cadmium showed significant non‐significant  interaction  was  found  between  PGPRs  and  RNPKF  at  2  mg  Cd  kg −1   soil,  but  the  non‐significant  interaction  was  found  between  PGPRs  and  RNPKF  at  2  mg  Cd  kg   soil,  but  the  −1 but negative correlation (0.6399; p = 0.0001) with FL. Inoculation of PGPRs (0.2239; p = 0.1893) gave interaction was non‐significant ordinal at 5 mg Cd kg−1  soil for FL. Cadmium showed significant but  interaction was non‐significant ordinal at 5 mg Cd kg  soil for FL. Cadmium showed significant but  non-significant positive correlation, while RNPKF (0.3835; p = 0.021) showed positive significant negative correlation (−0.6399; p = 0.0001) with FL. Inoculation of PGPRs (0.2239; p = 0.1893) gave non‐ negative correlation (−0.6399; p = 0.0001) with FL. Inoculation of PGPRs (0.2239; p = 0.1893) gave non‐ Environments 2020, 7, x; doi: FOR PEER REVIEW  6 of 16  Environments 2020, 7, x; doi: FOR PEER REVIEW  6 of 16  Environments 2020, 7, 54 6 of 16 significant  positive  correlation,  while  RNPKF  (0.3835;  p  =  0.021)  showed  positive  significant  correlation with FL (Figure 5). The maximum increase of 19 and 29% in FL was observed from control  significant  positive  correlation,  while  RNPKF  (0.3835;  p  =  0.021)  showed  positive  significant  −1 corr where elation  RNPK with F +FL A.(Figur  fabrum e 5was ). The  applied maximum  at 2 and incr 5ease  mg of Cd 19 kg and soil, 29% rein spectively. FL was obse   rved from control correlation with FL (Figure 5). The maximum increase of 19 and 29% in FL was observed from control  −1 where RNPKF + A. fabrum was applied at 2 and 5 mg Cd kg soil, respectively. where RNPKF + A. fabrum was applied at 2 and 5 mg Cd kg  soil, respectively.  2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil 2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil ab abc abcd a abcd abcd ab abc bcd cd de de de abcd abcd abcd bcd cd de 6 de de Control RNPKF S. A. fabrum RNPKF + S. RNPKF + A. maltophilia maltophilia fabrum Control RNPKF S. A. fabrum RNPKF + S. RNPKF + A. Treatments maltophilia maltophilia fabrum Treatments Figure 4. Bitter gourd fruit length (cm) treated with PGPRs, RNPKF, and their combination under 2  −1 Figure 4. Bitter gourd fruit length (cm) treated with PGPRs, RNPKF, and their combination under 2 and 5 mg Cd kg  soil. Different small letters express significant differences at p ≤ 0.05.  Figure 4. Bitter gourd fruit length (cm) treated with PGPRs, RNPKF, and their combination under 2  and 5 mg Cd kg −1 soil. Di erent small letters express significant di erences at p  0.05. and 5 mg Cd kg  soil. Different small letters express significant differences at p ≤ 0.05.  Fruit Length Fruit Length 0.60 0.60 0.3835* 0.40 0.3835* 0.2239ns 0.40 0.20 0.2239ns 0.021 0.0001 0.1893 0.20 0.00 0.021 0.0001 0.1893 Cd PGPR RNPKF 0.00 -0.20 Cd PGPR RNPKF -0.20 -0.40 -0.40 -0.60 -0.6399* -0.60 -0.80 -0.6399* -0.80 Figure 5. Pearson correlation of Cd, PGPRs, and RNPKF for fruit length (FL). * = significant (p  0.05); Figure 5. Pearson correlation of Cd, PGPRs, and RNPKF for fruit length (FL). * = significant (p ≤ 0.05);  ns = non-significant. ns = non‐significant.  Figure 5. Pearson correlation of Cd, PGPRs, and RNPKF for fruit length (FL). * = significant (p ≤ 0.05);  3.3. Bitter Gourd Yield per Plant ns = non‐significant.  3.3. Bitter Gourd Yield per Plant  PGPRs S. maltophilia and A. fabrum and RNPKF under 2 and 5 mg Cd kg soil significantly 3.3. Bitter Gourd Yield per Plant  −1 PGPRs S. maltophilia and A. fabrum and RNPKF under 2 and 5 mg Cd kg  soil significantly (p ≤  (p  0.05) a ect bitter gourd yield per plant (YP). At 2 mg Cd kg soil, inoculation of PGPRs has −1 −1 0.05) affect bitter gourd yield per plant (YP). At 2 mg Cd kg  soil, inoculation of PGPRs has significant  significant PGPRs main  S. mal and toph interactive ilia and A. e  fabrum ects on and YP. RN Application PKF unde of r 2 RNPKF  and 5 mg has Cd a significant  kg  soil simain gnificeant ect ly on (p ≤  −1 main and interactive effects on YP. Application of RNPKF has a significant main effect on YP at 5 mg  0.05) affect bitter gourd yield per plant (YP). At 2 mg Cd kg  soil, inoculation of PGPRs has significant  YP at 5 mg Cd kg soil. Treatment RNPKF + A. fabrum was significantly di erent as compared to −1 Cd kg  soil. Treatment RNPKF + A. fabrum was significantly different as compared to control at 2 and  contr main ol and at 2 int and eract 5 mg iveCd  effe kg cts on soil YP. Cd Applica for YP t(Figur ion ofe RNP 6). ItKwas F has observed  a signific that ant the main interaction  effect on YP of PGPRs  at 5 mg  −1 −1 5 mg Cd kg  soil Cd for YP (Figure 6). It was observed that the interaction of PGPRs and RNPKF was  Cd kg  soil. Treatment RNPKF + A. fabrum was significantly different as compared to control at 2 and  and RNPKF was significant ordinal at 2 mg Cd kg soil (Figure 7A) but non-significant ordinal at −1 −1 1−1 significant ordinal at 2 mg Cd kg  soil (Figure 7A) but non‐significant ordinal at 5 mg Cd kg  soil for  55 mg  mg Cd Cd kg kg soil  soilfor  CdYP  for(Figur  YP (Feigur 7B).e Heavy 6). It was metal  observed Cd showed  that the significant  interaction negative  of PGP corr Rs  elation and RN (PK 0.4385; F was  −1 −1 YP (Figure 7B). Heavy metal Cd showed significant negative correlation (−0.4385; p = 0.0075) with YP.  significant ordinal at 2 mg Cd kg  soil (Figure 7A) but non‐significant ordinal at 5 mg Cd kg  soil for  p = 0.0075) with YP. However, PGPRs (0.5035; p = 0.0017) and RNPKF (0.3829; p = 0.0212) showed YP (Figure 7B). Heavy metal Cd showed significant negative correlation (−0.4385; p = 0.0075) with YP.  Bittergourd Fruit Length (cm) Bittergourd Fruit Length (cm) Environments 2020, 7, x; doi: FOR PEER REVIEW  7 of 16  Environments 2020, 7, x; doi: FOR PEER REVIEW  7 of 16  Environments 2020, 7, 54 7 of 16 However, However, PG  PGPRs PRs ( 0(.05.03 503 5;5; p  p=  =0. 0. 0000 171)7 )and  and RNPKF  RNPKF (0 (0 .3829 .3829 ; p;  =p  0. = 02 0.02 121) 2showe ) showe d positive d positive  signif  signif icant icant     positive correlation correlation significant  wi withth YP  YP corr  (F (F igu elation igu rer e8) 8) .with  The . The YP ma ma (Figur ximum ximum e 8incr ). incr The eaea se maximum s of e of 26 26 .5 .5 and  incr and 21 ease  21 .1% .1%  of in  26.5 in YP YP  was and  was  observed 21.1%  observed in YP  from  was from     −1 −1 observed control, control, wher  wher frome e contr RNPKF  RNPKF ol, wher +  +A. A. fabrum e fabrum RNPKF  wa wa s + asA. pplied applied fabrum at at was 2  an 2 an d applied  d 5  mg 5 mg Cd at Cd 2kg and  kg soil, 5 soil, mg  respect  Cd respect kgively ively soil, .  . respectively. 2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil 2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil 0.9 0.9 0.8 b 0.8 b bc bcd bc bcd cd cd cd cde cd cde 0.7 cde cde cde 0.7 cde cde de cde de 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0.0 0.0 Control RNPKF S. A. fabrum RNPKF + S. RNPKF + Control RNPKF S. A. fabrum RNPKF + S. RNPKF + maltophilia maltophilia A. fabrum maltophilia maltophilia A. fabrum Treatments Treatments Figure 6. Bitter gourd yield (kg) per plant treated with PGPRs, RNPKF, and their combination under  Figure 6. Bitter gourd yield (kg) per plant treated with PGPRs, RNPKF, and their combination under 2 Figure 6. Bitter gourd yield (kg) per plant treated with PGPRs, RNPKF, and their combination under  −1 2 and 5 mg Cd kg  soil. 1  Different small letters express significant differences at p ≤ 0.05.  and 5 mg Cd kg−1 soil. Di erent small letters express significant di erences at p  0.05. 2 and 5 mg Cd kg  soil. Different small letters express significant differences at p ≤ 0.05.  −1 −1 Estimated Marginal Means of YP 2 mg Cd kg  soil  Estimated Marginal Means of YP 5 mg Cd kg  soil  −1 −1 Estimated Marginal Means of YP 2 mg Cd kg  soil  Estimated Marginal Means of YP 5 mg Cd kg  soil        (A)  (B)  (A)  (B)  p < 0.05  p > 0.05  p < 0.05  p > 0.05  Figur Figure e 7. Interaction 7. Interaction  graph graph  of S. maltoph of S. maltophilia ilia (NS1), (NS1),  A. fabrA. umfabrum  (NS2), (NS2),  and RNPKF and RNPKF  at 2 (A at) and 2 (A 5 ) mg and Cd  Figur −1 e 7. Interaction graph of S. maltophilia (NS1), A. fabrum (NS2), and RNPKF at 2 (A) and 5 mg Cd  kg  so 5 mg il (B Cd ) for kg bitter soil go (B)ur ford bitter yield gour per d pla yield nt (YP per).plant   (YP). −1 kg  soil (B) for bitter gourd yield per plant (YP).  Yield per Plant (kg / plant) Yield per Plant (kg / plant) Environments 2020, 7, x; doi: FOR PEER REVIEW  8 of 16  Environments 2020, 7, 54 8 of 16 Environments 2020, 7, x; doi: FOR PEER REVIEW  8 of 16  Yield per Plant Yield per Plant 0.60 0.5035* 0.60 0.5035* 0.3829* 0.40 0.3829* 0.40 0.20 0.20 0.0075 0.0212 0.0017 0.0075 0.0212 0.00 0.0017 0.00 Cd PGPR RNPKF Cd PGPR RNPKF -0.20 -0.20 -0.40 -0.40 ̶ 0.4385* ̶ 0.4385* -0.60 -0.60 Figure 8. Pearson correlation of Cd, PGPRs, and RNPKF for yield per plant (YP)* = significant (p ≤  Figure 8. Pearson correlation of Cd, PGPRs, and RNPKF for yield per plant (YP)* = significant (p  0.05); Figure 8. Pearson correlation of Cd, PGPRs, and RNPKF for yield per plant (YP)* = significant (p ≤  0.05); ns = non‐significant.  ns = non-significant. 0.05); ns = non‐significant.  3.4. Nitrogen Concentration in Bitter Gourd 3.4. Nitrogen Concentration in Bitter Gourd   3.4. Nitrogen Concentration in Bitter Gourd   PGPRs and RNPKF significantly (p  0.05) changed the nitrogen concentration of bitter gourd PGPRs and RNPKF significantly (p ≤ 0.05) changed the nitrogen concentration of bitter gourd  (NB) under di erent levels of Cd. Main e ects of PGPRs and RNPKF were significant on NB at 2 (NB)PGPRs  under  d and iffer RNPKF ent level signif s of Cd. ica nMai tly n( pe ≤ ffect  0.05 s of )  ch PGP ange Rsd and  the RN  nitrogen PKF were  concentration  significant  on of  NB bitter  at  2go and urd    −1 and 5 mg kg soil. However, the interaction of PGPRs and RNPKF was non-significant, ordinal at 2 (NB 5 mg ) und  kg e rsoil.  differ  However, ent level the s of  int Cd. eract  Maiion n e of ffect  PGP s ofR sPGP  andR RN s and PKF RN was PK Fnon  were ‐signif  sign ica ifica nt, nordin t on NB al at at 2  2and  and    −1 −1 and 5 mg Cd kg soil for NB. It was observed that RNPKF + S. maltophilia and RNPKF + A. fabrum 55 mg  mg kg  Cd  kg soil.  soil However,  for NB. the  It was  inte ract observed ion of  tha PGP t RNPK Rs andF  RN + S.PKF  maltop  was hilia non  and‐si gnif RNPica KFn t,+  ordin A. fabrum al at  were 2 and    −1 −1 wer sign e if significantly icantly different di er as ent coas mpa compar red to ed con totro contr l at 2 ol an atd 25 and mg Cd 5 mg  kg Cd soil kg for soil NB (Fi forgNB ure (Figur 9). Heavy e 9).  5 mg Cd kg  soil for NB. It was observed that RNPKF + S. maltophilia and RNPKF + A. fabrum were  −1 Heavy metal Cd showed significant negative correlation (0.4812; p = 0.0030) with NB. However, PGPRs sign meta ificant l Cd lsho y different wed signific  as coant mpa  negative red to  co conrrelation trol at 2  (an−0.48 d 512 mg ; p  Cd = 0.00  kg30) soil  with  for NB  NB.  However, (Figure 9) .PGP  Heavy Rs    (0.4391; (0.4391;p =p 0.0074 =  0.00)7showed 4)  showsignificant ed  significand ant  and RNPKF   RNPKF (0.2041;   (0.204 p =1; 0.2324)   p  =  0.232 showed 4)  sho non-significant wed  non‐signi positive ficant  metal Cd showed significant negative correlation (−0.4812; p = 0.0030) with NB. However, PGPRs  corr positive elation cor with relation NB (Figur with NB e 10 (F ).igure The 10 maximum ). The maximum increase incre of 48 ase and  of 48 56%  and in 56 NB % in was  NBobserved  was observed from  (0.4391;  p  =  0.0074)  showed  significant  and  RNPKF  (0.2041;  p  =  0.2324)  showed  non‐significant  −1 from control where RNPKF + S. maltophilia was applied at 2 and 5 mg Cd kg  soil, respectively.  control where RNPKF + S. maltophilia was applied at 2 and 5 mg Cd kg soil, respectively. positive correlation with NB (Figure 10). The maximum increase of 48 and 56% in NB was observed  −1 from control where RNPKF + S. maltophilia was applied at 2 and 5 mg Cd kg  soil, respectively.  2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil 2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil 4.5 ab 4.0 4.5 abc a abcd ab cd bcd cd 3.5 4.0 abc abcd de cd cde bcdcde cd 3.0 3.5 de 2.5 cde cde 3.0 2.0 2.5 1.5 2.0 1.0 1.5 0.5 1.0 0.0 0.5 Control RNPKF S. A. fabrum RNPKF + S. RNPKF + A. 0.0 maltophilia maltophilia fabrum Control RNPKF S. A. fabrum RNPKF + S. RNPKF + A. Figure 9. Nitrogen concentration in bitter maltophilia gourd (%) treated with PGPRs, RNPKF maltophilia , and their combination fabrum Figure  9.  Nitrogen  concentration  in  bitter  gourd  (%)  treated  with  PGPRs,  RNPKF,  and  their  under 2 and 5 mg Cd kg soil. Di erent small letters express significant di erences (p  0.05). −1 combination under 2 and 5 mg Cd kg  soil. Different small letters express significant differences (p  Figure  9.  Nitrogen  concentration  in  bitter  gourd  (%)  treated  with  PGPRs,  RNPKF,  and  their  ≤ 0.05).  −1 combination under 2 and 5 mg Cd kg  soil. Different small letters express significant differences (p  ≤ 0.05).  Nitrogen in Bittergourd (%) Nitrogen in Bittergourd (%) Environments 2020, 7, x; doi: FOR PEER REVIEW  9 of 16  Environments 2020, 7, 54 9 of 16 Environments 2020, 7, x; doi: FOR PEER REVIEW  9 of 16  Nitrogen concentration 0.60 Nitrogen concentration 0.4391* 0.60 0.40 0.4391* 0.2041ns 0.40 0.20 0.2324 0.2041ns 0.0074 0.003 0.20 0.00 0.2324 Cd PGPR RNPKF 0.0074 0.003 0.00 -0.20 Cd PGPR RNPKF -0.20 -0.40 ̶ 0.4812* -0.40 -0.60 ̶ 0.4812* -0.60 Figure 10. Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd nitrogen concentration  (NB) * = significant (p ≤ 0.05); ns = non‐significant.  Figure 10. Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd nitrogen concentration (NB) Figure 10. Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd nitrogen concentration  * = significant (p  0.05); ns = non-significant. 3.5. Phosphorus Concentration in Bitter Gourd   (NB) * = significant (p ≤ 0.05); ns = non‐significant.  3.5. Phosphorus Concentration in Bitter Gourd −1 Effect of PGPRs and RNPKF under 2 and 5 mg kg  soil was significant (p ≤ 0.05) on phosphorus  3.5. Phosphorus Concentration in Bitter Gourd   E ect of PGPRs and RNPKF under 2 and 5 mg kg soil was significant (p  0.05) on phosphorus concentration of bitter gourd (PB). Treatments RNPKF, RNPKF + S. maltophilia, RNPKF + A. fabrum,  −1 −1 concentration and RNPKF differed of bitter signific gourdantly (PB). at Tr 5 eatments  mg Cd kg RNPKF  soil over , RNPKF  contr+olS. for maltophilia  PB (Figur ,e RNPKF  11). App +lication A. fabrum,  of  Effect of PGPRs and RNPKF under 2 and 5 mg kg  soil was significant (p ≤ 0.05) on phosphorus  −1 −1 and RNPKF di ered significantly at 5 mg Cd kg soil over control for PB (Figure 11). Application of concentration RNPKF and PGPRs  of bitter  show  gourd ed a  (PB sign).if Treat icantm ma ents in  eRN ffect PK on F,  PB RNPKF  at 2 mg +  S. Cd maltop  kg  soil. hilia At , RN  5 mg PKF Cd +  A. kg fa bsoil, rum ,  1 1 −1 RNPKF and PGPRs  RNPK  and and FPGPRs   differed RNPKF showed   signific have aa antly signific significant  atant  5 mg  ma main  Cd in  ekg and ect  soi in on tel ract over PB at ive contr 2 ef mg fec oCd lt  for on kg  PB PB. (Figu soil.  Ordinal At re 11 5  mg int ). App eCd ractlkg ication ion wa soil,  sof    −1 −1 −1 PGPRs found and between RNPKF   PGP have Rs  aan significant d  RNPKFmain   at  2 and mg  interactive Cd  kg   soeil   but ect on signi PB.fica Orn dinal t  ord interaction inal  interact was ionfound   was  RNPKF and PGPRs showed a significant main effect on PB at 2 mg Cd kg  soil. At 5 mg Cd kg  soil,  −1 observed  at  5  mg  Cd  kg   soil  (Figure  12A,B)  for  PB.  Cadmium  showed  a  significant  negative  between PGPRs and PGPRs  RNPKF and RNPKF have a  at signific 2 mgant Cd ma kg in soil andbut  inte significant ractive effec ordinal t on PB. interaction  Ordinalwas  inteobserved raction wa ats  −1 5correlation mg Cd kg (−0. soil 661 (Figur 4; p = e0.00 12A,B) 01) with for PB. BDP. Cadmium  However, showed  PGPR (a0.253 significant 7; p = 0.19 negative 53) showed corr elation non‐signi (0.6614; ficant  found  between  PGPRs  and  RNPKF  at  2  mg  Cd  kg   soil  but  significant  ordinal  interaction  was  −1 and  RNPKF  (0.4422;  p  =  0.0069)  showed  significant  positive  correlation  with  PB  (Figure  13).  The  pobserved = 0.0001)  at with   5  mg BDP  Cd . However   kg   soil , PGPR   (Figure (0.2537;   12A,B) p =  0.1953) for  PB.showed   Cadmiu non-significant m  showed  a  signific and RNPKF ant  negativ (0.4422;e  maximum increase of 29.5% in PB was observed from control where RNPKF + A. fabrum was applied  p = 0.0069) showed significant positive correlation with PB (Figure 13). The maximum increase of correlation (−0.6614; p = 0.0001) with BDP. However, PGPR (0.2537; p = 0.1953) showed non‐significant  −1 1 at 2 mg Cd kg  soil.  29.5% and  RN inPKF PB was   (0.442 observed 2;  p  =  0.00 from69) contr   showe ol wher d  signific e RNPKF ant  positive + A. fabrum   corr was elation applied   with at  PB 2 mg   (Fig Cd ure kg  13).soil.   The  maximum increase of 29.5% in PB was observed from control where RNPKF + A. fabrum was applied  −1 at 2 mg Cd kg  soil.  2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil 1.0 2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil ab a 0.8 1.0 abc bcd bcd cd cd cd cd ab a de 0.6 0.8 abc bcd bcd cd cd cd cd de 0.4 0.6 0.2 0.4 0.0 0.2 Control RNPKF S. A. fabrum RNPKF + S. RNPKF + maltophilia maltophilia A. fabrum 0.0 Figure 11. Phosphorus concentration in bitter gourd (%) treated with PGPRs, RNPKF, and their Control RNPKF S. A. fabrum RNPKF + S. RNPKF + Figure  11.  Phosphorus  concentration  in  bitter  gourd  (%)  treated  with  PGPRs,  RNPKF,  and  their  combination under 2 and 5 mg Cd kg soil. Di erent small letters express significant di erences −1 maltophilia maltophilia A. fabrum combination under 2 and 5 mg Cd kg  soil. Different small letters express significant differences (p  (p  0.05). ≤ 0.05).  Figure  11.  Phosphorus  concentration  in  bitter  gourd  (%)  treated  with  PGPRs,  RNPKF,  and  their  −1 combination under 2 and 5 mg Cd kg  soil. Different small letters express significant differences (p  ≤ 0.05).  Phosphorus in Bittergourd Phosphorus in Bittergourd -1 -1 (mg kg ) (mg kg ) Environments 2020, 7, x; doi: FOR PEER REVIEW  10 of 16  Environments 2020, 7, 54 10 of 16 Environments 2020, 7, x; doi: FOR PEER REVIEW  10 of 16  −1 −1 Estimated Marginal Means of PB 2 mg Cd kg  soil  Estimated Marginal Means of PB 5 mg Cd kg  soil  −1 −1 Estimated Marginal Means of PB 2 mg Cd kg  soil  Estimated Marginal Means of PB 5 mg Cd kg  soil     (A)  (B)     p > 0.05  p < 0.05  (A)  (B)  p > 0.05  p < 0.05  Figure 12. Interaction graphs of S. maltophilia (NS1), A. fabrum (NS2), and RNPKF at 2 (A) and 5 mg  −1 Cd kg  soil (B) for phosphorus concentration in bitter gourd (PB).  Figure 12. Interaction graphs of S. maltophilia (NS1), A. fabrum (NS2), and RNPKF at 2 (A) and 5 mg  Figure 12. Interaction graphs of S. maltophilia (NS1), A. fabrum (NS2), and RNPKF at 2 (A) and −1 5 mg Cd kg soil (B) for phosphorus concentration in bitter gourd (PB). Cd kg  soil (B) for phosphorus concentration in bitter gourd (PB).  Phosphorus concentration Phosphorus concentration 0.60 0.4422* 0.60 0.40 0.4422* 0.1953ns 0.40 0.1953ns 0.20 0.0069 0.2537 0.0001 0.20 0.00 0.0069 0.2537 0.0001 Cd PGPR RNPKF 0.00 -0.20 Cd PGPR RNPKF -0.20 -0.40 -0.40 -0.60 -0.60 ̶ 0.6614* -0.80 ̶ 0.6614* -0.80 Figure 13. Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd phosphorus concentration Figure 13. Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd phosphorus concentration  (NB). * = significant (p  0.05); ns = non-significant. (NB). * = significant (p ≤ 0.05); ns = non‐significant.  Figure 13. Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd phosphorus concentration  3.6. Potassium Concentration in Bitter Gourd (NB). * = significant (p ≤ 0.05); ns = non‐significant.  3.6. Potassium Concentration in Bitter Gourd   Influence of PGPRs and RNPKF 2 and 5 mg kg soil was significant (p  0.05) on potassium 3.6. Potassium Concentration in Bitter Gourd   concentration of bitter gourd (KB). It was also observed −1 that RNPKF + S. maltophilia and RNPKF + Influence of PGPRs and RNPKF 2 and 5 mg kg  soil was significant (p ≤ 0.05) on potassium  A. fabrum di ered significantly for KB at 5 mg Cd kg soil (Figure 14). Both PGPRs and RNPKF have −1 concentration of bitter gourd (KB). It was also observed that RNPKF + S. maltophilia and RNPKF + A.  Influence of PGPRs and RNPKF 2 and 5 mg kg  soil was significant (p ≤ 0.05) on potassium  −1 a significant main e ect on KB at 2 and 5 mg Cd kg soil. Disordinal non-significant interaction was fabrum differed significantly for KB at 5 mg Cd kg  soil (Figure 14). Both PGPRs and RNPKF have a  concentration of bitter gourd (KB). It was also observed that RNPKF + S. maltophilia and RNPKF + A.  found between PGPRs and RNPKF at 2 mg Cd kg−1 soil and but ordinal interaction was observed significant main effect on KB at 2 and 5 mg Cd kg −1  soil. Disordinal non‐significant interaction was  fabrum differed significantly for KB at 5 mg Cd kg  soil (Figure 14). Both PGPRs and RNPKF have a  −1 at 5 mg Cd kg soil for KB. Di erent levels of Cd showed significant negative correlation (0.4904; found between PGPRs and RNPKF at 2 mg Cd kg−1 soil and but ordinal interaction was observed at  significant main effect on KB at 2 and 5 mg Cd kg  soil. Disordinal non‐significant interaction was  p = 0.0024) with −1 KB. However, PGPR (0.5516; p = 0.0005) and RNPKF (0.3840; p = 0.0208) showed 5 mg Cd kg  soil for KB. Different levels of Cd showe −1 d significant negative correlation (−0.4904; p =  found between PGPRs and RNPKF at 2 mg Cd kg  soil and but ordinal interaction was observed at  significant positive correlation with KB (Figure 15). Application of RNPKF + S. maltophilia, RNPKF 0.0024)  with −1  KB.  However,  PGPR  (0.5516;  p  =  0.0005)  and  RNPKF  (0.3840;  p  =  0.0208)  showed  5 mg Cd kg  soil for KB. Different levels of Cd showed significant negative correlation (−0.4904; p =  + A. fabrum, RNPKF, S. maltophilia and A. fabrum were significantly di erent as compared to control significant positive correlation with KB (Figure 15). Application of RNPKF + S. maltophilia, RNPKF +  0.0024)  with  KB.  However,  PGPR  (0.5516;  p  =  0.0005)  and  RNPKF  (0.3840;  p  =  0.0208)  showed  at 2 mg Cd kg soil for KB. The maximum increase of 72 and 55% in KB was observed from control A. fabrum, RNPKF, S. maltophilia and A. fabrum were significantly different as compared to control at  significant positive correlation with KB (Figure 15). Application of RNPKF + S. maltophilia, RNPKF +  where RNPKF + A. fabrum was applied at 2 and 5 mg Cd kg soil, respectively. A. fabrum, RNPKF, S. maltophilia and A. fabrum were significantly different as compared to control at  Environments 2020, 7, x; doi: FOR PEER REVIEW  11 of 16  −1 2 mg Cd kg  soil for KB. The maximum increase of 72 and 55% in KB was observed from control  −1 where RNPKF + A. fabrum was applied at 2 and 5 mg Cd kg  soil, respectively.  Environments 2020, 7, x; doi: FOR PEER REVIEW  11 of 16  Environments 2020, 7, 54 2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil 11 of 16 −1 2 mg Cd kg  soil for KB. The maximum increase of 72 and 55% in KB was observed from control  −1 9.0 where RNPKF + A. fabrum was applied at 2 and 5 mg Cd kg  soil, respectively.  ab 2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil 7.5 bc bcd bcd bcde cde 9.0 6.0 cdef def ef ab 7.5 bc bcd 4.5 bcd bcde cde 6.0 cdef def ef 3.0 4.5 1.5 3.0 0.0 1.5 Control RNPKF S. maltophilia A. fabrum RNPKF + S. RNPKF + A. maltophilia fabrum 0.0 Control RNPKF S. maltophilia A. fabrum RNPKF + S. RNPKF + A.   maltophilia fabrum Figure  14.  Potassium  concentration  in  bitter  gourd  (%)  treated  with  PGPRs,  RNPKF,  and  their  −1   combinat Figureion 14. under Potassium  2 and concentration 5 mg Cd kg in soil. bitter  Different gourd  (%) small treated  letters with  express PGPRs,  significant RNPKF, and differen theirces (p  combination under 2 and 5 mg Cd kg soil. Di erent small letters express significant di erences ≤ 0.05).  Figure  14.  Potassium  concentration  in  bitter  gourd  (%)  treated  with  PGPRs,  RNPKF,  and  their  (p  0.05). −1 combination under 2 and 5 mg Cd kg  soil. Different small letters express significant differences (p  ≤ 0.05).  Potassium concentration 0.80 Potassium concentration 0.5516* 0.80 0.60 0.384* 0.5516* 0.60 0.40 0.384* 0.40 0.20 0.0208 0.0024 0.0005 0.20 0.00 0.0208 0.0024 0.0005 Cd PGPR RNPKF 0.00 -0.20 Cd PGPR RNPKF -0.20 -0.40 -0.40 ̶ 0.4904* -0.60 ̶ 0.4904* -0.60 Figure 15. Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd potassium concentration Figure 15. Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd potassium concentration  (NB). * = significant (p  0.05); ns = non-significant. Figure 15. Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd potassium concentration  (NB). * = significant (p ≤ 0.05); ns = non‐significant.  4. Discussion (NB). * = significant (p ≤ 0.05); ns = non‐significant.  4. Discussion  A significant decrease in fruit length, fresh weight, and yield per plant of bitter gourd were 4. Discussion  observed in control at 5 mg Cd kg soil. Low uptake of N, P, and K in bitter gourd under Cd toxicity A significant decrease in fruit length, fresh weight, and yield per plant of bitter gourd were  A significant decrease in fruit length, fresh weight, and yield per plant of bitter gourd were  −1 might be a major factor for reduction in yield, fruit length, and fresh weight. Higher biosynthesis observed in control at 5 mg Cd kg  soil. Low uptake of N, P, and K in bitter gourd under Cd toxicity  −1 observed in control at 5 mg Cd kg  soil. Low uptake of N, P, and K in bitter gourd under Cd toxicity  of stress ethylene might be an allied factor responsible for a significant decline in yield of bitter might be a major factor for reduction in yield, fruit length, and fresh weight. Higher biosynthesis of  might be a major factor for reduction in yield, fruit length, and fresh weight. Higher biosynthesis of  gourd under Cd stress. According to Sanita di Toppi and Gabbrielli [7], accumulation of Cd beyond stress ethylene might be an allied factor responsible for a significant decline in yield of bitter gourd  stress ethylene might be an allied factor responsible for a significant decline in yield of bitter gourd  safe limit disturbed the nutrients homeostasis which played an imperative role in reduction of root under Cd stress. According to Sanita di Toppi and Gabbrielli [7], accumulation of Cd beyond safe  under Cd stress. According to Sanita di Toppi and Gabbrielli [7], accumulation of Cd beyond safe  and shoot elongation. Cadmium in plants also competes with divalent nutrients ions and decreases limit disturbed the nutrients homeostasis which played an imperative role in reduction of root and  limit disturbed the nutrients homeostasis which played an imperative role in reduction of root and  their uptake in plants [16]. Under Cd toxicity, transmembrane carriers in roots become unable to shoot elongation. Cadmium in plants also competes with divalent nutrients ions and decreases their  shoot elongation. Cadmium in plants also competes with divalent nutrients ions and decreases their  distinguish between non-essential Cd and essential divalent nutrients during their uptake [53,54]. uptake  in  plants  [16].  Under  Cd  toxicity,  transmembrane  carriers  in  roots  become  unable  to  uptake  in  plants  [16].  Under  Cd  toxicity,  transmembrane  carriers  in  roots  become  unable  to  Glick et al. [55] also documented that biosynthesis of endogenous stress ethylene under abiotic stress conditions, negatively a ects the productivity of the crop. Toxicity of heavy metals causes abnormal division of cell thus induced chromosomal aberration in plants [56]. This resulted in a decrease of protochlorophyllide reductase activity. Such disturbance in plants also induced chlorosis in leaves [57]. -1 -1 Potassium in Bittergourd (mg kg ) Potassium in Bittergourd (mg kg ) Environments 2020, 7, 54 12 of 16 Furthermore, Matile et al. [58] suggested the decomposition of lipids in cell wall when ethylene concentration is increased. They argued that ethylene when contact with chlorophyllase (chlase) gene it degrades chlorophyll caused in chlorosis. Furthermore, application of RNPKF + A. fabrum di ered significantly better from the sole application of control for improvement in N, P and K. The improvement in N, P, and K mitigate the adverse impacts of Cd in bitter gourd. Pankovic ´ et al. [59] observed that improvement in N uptake of sunflower alleviants the inhibitory influences of Cd [22,23,27,28,32]. Higher N facilitates in activity of Rubisco by an increase in soluble protein contents. Application of N in NH form is ecacious in decreasing the Cd uptake due to antagonistic relationship [60]. Findings of the current experiment also support the above argument. Better N in bitter gourd was observed where yield was improved over control even under Cd toxicity. Under Cd stress, plants start producing N metabolites, i.e., proline that causes phytochelation and decreases the intake of Cd [61]. Application of phosphorus neutralizes the adverse impacts of Cd and improve the yield of crops [62]. Improvement of P uptake in plants enhances the synthesis of glutathione that prevents membrane damages caused by Cd [63]. Balance K concentration decreases the generation of reactive oxidative species (ROS) and inhibits the NADPH oxidase [64]. Moreover, less generation of stress ethylene by inoculation of A. fabrum and RNPKF + A. fabrum might be another major factor responsible for the enhancement in bitter gourd growth and yield in the current study. Both PGPRs were capable to produce ACC deaminase, which cleaves ethylene into intermediate compounds. Similar kinds of results were also documented by many scientists [25,26,30,31]. Glick et al. [44] proposed that enzyme ACC deaminase break ethylene into -ketobutyrate and ammonia [65,66]. Accumulated ethylene in roots moved towards rhizosphere; thus, ethylene becomes low in plant roots, and stress is alleviated. Similarly, Tripathi et al. [67] reported growth hormones, indole acetic acid, improved the root elongation for better uptake of nutrients [24]. 5. Conclusions It is concluded that PGPR, A. fabrum has more potential over S. maltophilia to alleviate Cd induced stress in bitter gourd. Inoculation of A. fabrum with RNPKF is an ecacious approach to improve N, P, and K concentration in bitter gourd. The combined use of RNPKF and A. fabrum can increase the number of bitter gourds per plant, bitter gourd fruit length, and yield per plant by alleviating 5 mg Cd kg soil induced toxicity. 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Alleviation of Cadmium Adverse Effects by Improving Nutrients Uptake in Bitter Gourd through Cadmium Tolerant Rhizobacteria

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environments Article Alleviation of Cadmium Adverse E ects by Improving Nutrients Uptake in Bitter Gourd through Cadmium Tolerant Rhizobacteria 1 1 1 , 2 , 3 , Muhammad Zafar-ul-Hye , Muhammad Naeem , Subhan Danish *, Shah Fahad * , 4 , 5 6 4 , 7 , 8 Rahul Datta * , Mazhar Abbas , Ashfaq Ahmad Rahi , Martin Brtnicky , 8 9 10 Jir ˇí Holátko , Zahid Hassan Tarar and Muhammad Nasir Department of Soil Science, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan 60000, Pakistan; zafarulhyegondal@yahoo.com (M.Z.-u.-H.); ch.naeem276@gmail.com (M.N.) Department of Agronomy, The University of Haripur, Haripur 22620, Pakistan College of Plant Sciences and Technology, Huazhong Agriculture University, Wuhan 430070, China Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, Faculty of AgriSciences, Mendel University in Brno, 61300 Brno, Czech Republic; martin.brtnicky@mendelu.cz Institute of Horticultural Sciences, Faculty of Agriculture, University of Agriculture, Faisalabad 38000, Pakistan; rmazhar@hotmail.com Pesticide Quality Control Laboratory, Multan 60000, Pakistan; rahisenior2005@gmail.com Institute of Chemistry and Technology of Environmental Protection, Brno University of Technology, Faculty of Chemistry, Purkynova 118, 62100 Brno, Czech Republic Department of Geology and Pedology, Faculty of Forestry and Wood Technology, Mendel University in Brno, 61300 Brno, Czech Republic; jiri.holatko@centrum.cz Soil and Water Testing Laboratory, Mandi Bahauddin 50400, Pakistan; zahidtarar123@yahoo.com Soil and Water Testing Laboratory for Research, Multan 60000, Pakistan; swt_mltn@yahoo.com * Correspondence: sd96850@gmail.com (S.D.); shah.fahad@mail.hzau.edu.cn (S.F.); rahulmedcure@gmail.com (R.D.) Received: 16 May 2020; Accepted: 22 July 2020; Published: 26 July 2020 Abstract: Cadmium is acute toxicity inducing heavy metal that significantly decreases the yield of crops. Due to high water solubility, it reaches the plant tissue and disturbs the uptake of macronutrients. Low uptake of nutrients in the presence of cadmium is a well-documented fact due to its antagonistic relationship with those nutrients, i.e., potassium. Furthermore, cadmium stressed plant produced a higher amount of endogenous stress ethylene, which induced negative e ects on yield. However, inoculation of 1-amino cyclopropane-1-carboxylate deaminase (ACCD), producing plant growth promoting rhizobacteria (PGPR), can catabolize this stress ethylene and immobilized heavy metals to mitigate cadmium adverse e ects. We conducted a study to examine the influence of ACCD PGPR on nutrients uptake and yield of bitter gourd under cadmium toxicity. Cadmium tolerant PGPRs, i.e., Stenotrophomonas maltophilia and Agrobacterium fabrum were inoculated solely and in combination with recommended nitrogen, phosphorus, and potassium fertilizers (RNPKF) applied under di erent concentration of soil cadmium (2 and 5 mg kg soil). Results showed that A. fabrum with RNPKF showed significant positive response towards an increase in the number of bitter gourds per plant (34% and 68%), fruit length (19% and 29%), bitter gourd yield (26.5% and 21.1%), N (48% and 56%), and K (72% and 55%) concentration from the control at di erent concentrations of soil cadmium (2 and 5 mg kg soil), respectively. In conclusion, we suggest that A. fabrum with RNPKF can more ecaciously enhance N, K, and yield of bitter gourd under cadmium toxicity. Keywords: ACC deaminase; heavy metal stress; PGPR; fertilizers; nutrients; yield Environments 2020, 7, 54; doi:10.3390/environments7080054 www.mdpi.com/journal/environments Environments 2020, 7, 54 2 of 16 1. Introduction High use of pesticides, inorganic fertilizers, and untreated sewage water has significantly contributed to the buildup of heavy metals in agricultural soils [1,2]. These heavy metals become part of soil at the exchange site and remain readily available for plants. Rapid industrialization and anthropogenic activities are also allied factors responsible for the accumulation of toxic metals beyond their threshold limit in cultivatable lands [3–6]. Among di erent heavy metals, cadmium (Cd) is an acute toxin due to its high resistance time, i.e., >1000 years and water solubility [7]. Presence of cadmium below 0.5 mg kg soil is considered a safe limit, but depending upon parent material, it can 1 1 be accumulated up to 3.0 mg kg soil [8]. Being a part of phosphate fertilizers (up to 4.4 mg kg ), it is easily taken up by crops as Cd-supplement [9,10]. Cadmium causes cardiovascular, respiratory, cancer, and renal, skeletal system in humans when taken up beyond the threshold limit [11,12]. The high concentration of Cd in plant tissues disturbs nutrient uptake and creates water imbalance that results in poor photosynthesis [13]. It also causes lipid membrane instability, alteration in membranes permeability, and chlorosis in plants [14,15]. Due to its divalent nature, it competes with divalent essential nutrients, i.e., P, Ca, Mg, and decreases their uptake in plants [16–18]. Bioavailability of K is also a ected when Cd is present in higher concentration in soil [19]. Heavy metal toxicity and physiochemical properties soil depend on the land use [20–22]. Di erent crops, the biological adsorption factor (mg Cd/kg plant ash, mg Cd/kg soil) based on Cd content in plant ash, is di erent, i.e., winter wheat grains (5.97) and straw (2.50), barley grains (4.06) and straw (2.50), sugar beet roots (4.63) and tops (1.41), pea beans (3.22) and straw (0.88), corn grains (8.75) and straw (2.53), soya beans (4.31) and straw (4.63), sunflower seeds (10.8) and stem (4.28) [23]. Moreover, biosynthesis of endogenous stress ethylene under Cd toxicity plays a notorious role that aids in poor root growth [24–26]. In addition, ethylene (C H ) is a plant-signaling molecule. It is involved in seed germination 2 4 flower senescence, root elongation, fruit ripening, and leaf abscission. Mostly ethylene is synthesized in a two-step process, i.e., (1) enzymatic conversion of S-adenosyl methionine (SAM) to 1-amino cyclopropane-1-carboxylic acid (ACC); (2) conversion of ACC to ethylene, which is catalyzed by ACC-oxidase [27]. However, synthesis of endogenous ethylene level is significantly enhanced upon exposure of plants to abiotic stresses, including low soil fertility [28,29]. This endogenous stress ethylene negatively a ects gas exchange attributes, nutrients and water uptake, and yield of di erent crops under any stress conditions [30,31]. To overcome these problems, inoculation of ACC deaminase producing plant growth promoting rhizobacteria (PGPR), could be an ecacious and nature friendly technique [32–36]. Certain PGPRs can improve growth attributes of crops under heavy metals toxic conditions by secretions of ACC deaminase, siderophores, indole acetic acid, gibberellins, and better availability of water and nutrients [37–40]. Enzyme ACC deaminase cleaves stress ethylene into intermediate compounds; thus, decreases the stress generating factors in plants [41,42]. Among di erent crop plants, bitter gourd is a rich source of vitamins, carbohydrates, and proteins [43,44]. As compared to cucumber and tomato, one cup of bitter gourd juice (94 g) has 93% reference daily intake (RDI) of vitamin C [45]. It is cultivated in Pakistan (6107 hectares), with an annual production of 57,190 tons [46]. However, the yield of bitter gourd is negatively a ected when cultivated in Cd pollution. As improvement in N, P, and K can mitigate the stress of Cd toxicity in plants [3], which is why the current study was conducted to explore the ecacy of ACC deaminase producing PGPR with recommended NPK fertilizers (RNPKF) on bitter gourd nutrients uptake and yield under Cd toxicity. Environments 2020, 7, 54 3 of 16 The present study aimed to explore (1) e ectiveness of rhizobacteria in the improvement of nutrients uptake; (2) e ect of nutrients on bitter gourd yield under cadmium-induced stress; (3) correlation of inorganic fertilizer with rhizobacteria on yield and nutrients attributes of bitter gourd under Cd stress. We hypothesized that ACC deaminase-producing PGPRs could improve nutrient uptake and alleviate adverse e ects of Cd in bitter gourd for yield improvement. 2. Materials and Methods 2.1. Experimental Site and Treatments A pot experiment was conducted in the Department of Soil Science research area, Bahauddin Zakariya University, Multan, Pakistan. The soil was characterized as dark brown and saline with JAKHAR soil series [42]. Six treatments were applied in four replication by following two factorial completely randomized designs (CRDs). The treatments were control (without NPK or bacterial strains), recommended NPK fertilizers (RNPKF), Stenotrophomonas maltophilia, Agrobacterium fabrum, RNPKF + S. maltophilia, and RNPKF + A. fabrum. All treatments were added in the soil at 2 and 5 mg Cd kg soil. Artificial toxicity of Cd was developed by using analytical grade salt of CdCl [25]. As per treatment plan, two levels of Cd were maintained, i.e., 2 and 5 ppm (mg kg soil), keeping in mind the Cd concentration of pre-experimental soil. Rhizobacteria were inoculated at the time of sowing. However, required fertilizers were applied at the time of pot preparation. 2.2. Collection of Bacterial Strains and Broth ACC deaminase producing PGPRs S. maltophilia (ACC deaminase activity = 71.78 mol 1 1 -ketobutyrate mg protein h ) and A. fabrum (ACC deaminase activity = 432.6 mol -ketobutyrate 1 1 mg protein h ) were taken from the Laboratory of Soil Microbiology, Department of Soil Science. Both PGPRs were documented Cd tolerant previously, i.e., survive over 5.0 mg Cd kg soil toxicity [25]. For seeds inoculation, Dworkin and Foster (DF) media without agar was used for inoculum preparation [47]. Loop of each rhizobacteria was taken in the sterilized flask and incubated at 25  3 C and 100 rpm for 48 h. After that, broth optical density (OD) was measured by spectrophotometer (540 nm wavelength). Finally, dilution was made with sterilized distilled water to 7 8 –1 achieve 0.45 nm OD, to achieve a uniform population of 10 –10 colony forming units (CFU) mL . 2.3. Seeds Sterilization and Sowing HgCl (0.1%) solution was used for sterilization of seeds. All seeds were placed for 5 min in the solution followed by, three times, washing with sterilized deionized water [48]. Moreover, 1mL respective PGPR inoculum was used for seeds inoculation along with sugar (30% sucrose), peat, and clay (1:1) in 1:2:6 ratio for 100 g seeds. Four inoculated seeds were sown in each pot. Sowing of bitter gourd seeds was done by hand. After 20 days of sowing, only three healthy seedlings were maintained in each pot by thinning. 2.4. Irrigation and Fertilizer Application In pots, 65% field capacity was maintained on a weight basis during the experiment. To fulfil the requirement of crop nutrients (187N, 75P, and 225K kg ha ) urea, K HPO and K SO were applied. 2 4 2 4 2.5. Harvesting and Samples Analyses Harvesting was done at the time of fruit maturity. Samples were digested for the determination of biochemical attributes. The number of bitter gourds was counted manually. For fruit length, standard measuring tape was used. For determination of yield per plant, fruits were collected and weighed on the analytical balance. With the help of diacid mixture nitric acid and perchloric acid (2:1 ratio), the tissues of the plant were digested for P and K analyses [49]. Phosphorus in the samples was determined by using ammonium molybdate and ammonium metavanadate yellow color method [50]. Environments 2020, 7, 54 4 of 16 However, for analyses of K in samples, the digested solution was run on flame photometer [51]. For determination of nitrogen, samples were digested in concentrated H SO , and digestion mixture 2 4 (K SO (100):CuSO .5H O (10):FeSO (1)). Distillation was performed in Kjeldahl distillation apparatus, 2 4 4 2 4 Environments 2020, 7, x; doi: FOR PEER REVIEW  4 of 16  using boric acid as a collector [52]. 2.6. Statistical Analyses  2.6. Statistical Analyses One‐way  ANOVA  was  used  to  assess  the  effects  of  treatments.  Two  factorial  ANOVA  was  One-way ANOVA was used to assess the e ects of treatments. Two factorial ANOVA was conducted  separately  to  compare  PGPRs  and  RNPK  interaction  under  different  levels  of  Cd.  conducted separately to compare PGPRs and RNPK interaction under di erent levels of Cd. Treatment comparison was computed at p ≤ 0.05 by Tukey’s Test.  Treatment comparison was computed at p  0.05 by Tukey’s Test. 3. Results  3. Results 3.1. Number of Bitter Gourds per Plant 3.1. Number of Bitter Gourds per Plant  E ects of PGPRs and RNPKF under di erent levels of Cd were significant (p  0.05) on the Effects  of PGPRs and  RNPKF under  different levels  of  Cd  were significant  (p ≤ 0.05)  on  the  number of bitter gourds per plants (BDP). Inoculation of PGPRs and RNPKF have significant main and number of bitter gourds per plants (BDP). Inoculation of PGPRs and RNPKF have significant main  −1 interactive and interact e iv ects e effect on BDP s on BDP at 2 and  at 2 5an mg d 5kg  mg kg soil. soil. Application  Application of RNPKF of RNPKF + S. + S. maltophilia  maltophilia , RNPKF , RNPKF +  A. fabrum, RNPKF, S. maltophilia and A. fabrum showed significant positive e ect over control at 2 and + A. fabrum, RNPKF, S. maltophilia and A. fabrum showed significant positive effect over control at 2  1 1 −1 −1 5and mg 5 Cd  mg kg Cd kg soil so foril BDP for BDP (Figur  (Fig eure 1). 1). Interaction  Interaction between  between PGPRs  PGPRs and  and RNPKF  RNPKF at at 2 2 mg  mg Cd  Cdkg kg  soil soil  −1 (Figur (Figure e 2 A) 2A) and  and 5 mg 5 mg Cd Cd kg kgsoil so (Figur il (Figeure 2B) 2B) wer were e significant  significant ordinal  ordinal for BDP  for BD (Figur P (F eig 2B). ureIt 2B) was . It noted  was  that noted Cd tha showed t Cd showed non-significant  non‐signnegative ificant negative correlation  correlation (0.1451;  (−0p.14 =51 0.3986) ; p = 0.with 3986)BDP  with . However  BDP. How , PGPR ever,  (0.5863; PGPR (p0.586 = 0.0002) 3; p = 0.000 and 2) RNPKF  and RN (0.3237; PKF (0p.3= 237 0.0541) ; p = 0.054 showed 1) showe positive d positive significant  significa corr nt elation  correlation with with BDP  BDP (Figure 3). The maximum increase of 34% and 68% in BDP was observed from control where  (Figure 3). The maximum increase of 34% and 68% in BDP was observed from control where RNPKF + 1 −1 A.RN fabrum PKF +was  A. fa applied brum wa ats 2ap and plied 5 mg at 2Cd  and kg 5 mg soil,  Cdr kg espectively  soil, respectively. .   2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil a a ab a a 16 ab ab ab bc Control RNPKF S. A. fabrum RNPKF + S. RNPKF + A. maltophilia maltophilia fabrum Treatments Figure 1. Number of bitter gourds per plant (BDP) treated with plant growth promoting rhizobacteria Figure 1. Number of bitter gourds per plant (BDP) treated with plant growth promoting rhizobacteria  (PGPRs), recommended NPK fertilizers (RNPKF), and their combination under 2 and 5 mg Cd kg −1 (PGPRs), recommended NPK fertilizers (RNPKF), and their combination under 2 and 5 mg Cd kg   soil. Di erent small letters express significant di erences (p  0.05). soil. Different small letters express significant differences (p ≤ 0.05).     No. of Bittergourds / Plant Environments 2020, 7, x; doi: FOR PEER REVIEW  5 of 16  Environments 2020, 7, 54 5 of 16 Environments 2020, 7, x; doi: FOR PEER REVIEW  5 of 16  −1 −1 Estimated Marginal Means of BDP 2 mg Cd kg  soil  Estimated Marginal Means of BDP 5 mg Cd kg  soil  −1 −1 Estimated Marginal Means of BDP 2 mg Cd kg  soil  Estimated Marginal Means of BDP 5 mg Cd kg  soil        (A)  (B)  (A)  (B)  p < 0.05  p < 0.05  p < 0.05  p < 0.05  Figure 2. Interaction graph of S. maltophilia (NS1), A. fabrum (NS2), and RNPKF at 2 (A) and 5 mg Cd  Figure −1  2. Interaction graph of S. maltophilia (NS1), A. fabrum (NS2), and RNPKF at 2 (A) and 5 mg Cd  Figure 2. Interaction graph of S. maltophilia (NS1), A. fabrum (NS2), and RNPKF at 2 (A) and kg  soil (B) for number of bitter gourd per plant (BDP).  −1 1 kg  soil (B) for number of bitter gourd per plant (BDP).  5 mg Cd kg soil (B) for number of bitter gourd per plant (BDP). Number of Bitter Gourd per Plant Number of Bitter Gourd per Plant 0.70 0.70 0.5863* 0.60 0.5863* 0.60 0.50 0.50 0.3986 0.40 0.3986 0.3237* 0.40 0.3237* 0.30 0.30 0.20 0.20 0.10 0.0541 0.0002 0.10 0.0541 0.0002 0.00 0.00 Cd PGPR RNPKF Cd PGPR RNPKF -0.10 -0.10 -0.20 -0.1451ns -0.20 -0.1451ns Figure 3. Pearson correlation of Cd, PGPRs, and RNPKF for number of bitter gourd per plant (BDP). Figure 3. Pearson correlation of Cd, PGPRs, and RNPKF for number of bitter gourd per plant (BDP).  * = significant (p  0.05); ns = non-significant. Figure 3. Pearson correlation of Cd, PGPRs, and RNPKF for number of bitter gourd per plant (BDP).  * = significant (p ≤ 0.05); ns = non‐significant.  * = significant (p ≤ 0.05); ns = non‐significant.  3.2. Bitter Gourd Fruit Length 3.2. Bitter Gourd Fruit Length  3.2. Bitte E ects r Gou ofrPGPRs d Fruit Len inoculation gth  and application of RNPKF under various Cd levels were significant (p  0.05) on bitter gourd fruit length (FL). Application of RNPKF + S. maltophilia and RNPKF + Effects of PGPRs inoculation and application of RNPKF under various Cd levels were significant  Effects of PGPRs inoculation and application of RNPKF under various Cd levels were significant  A. fabrum were significantly di erent from control at 2 and 5 mg Cd kg soil for FL. It was observed (p ≤ 0.05) on bitter gourd fruit length (FL). Application of RNPKF + S. maltophilia and RNPKF + A.  (p ≤ 0.05) on bitter gourd fruit length (FL). Application of RNPKF + S. maltophilia and RNPKF + A.  −1 that RNPKF showed a positive significantly better response at 2 mg Cd kg soil but remained fabrum were significantly different from control at 2 and 5 mg Cd kg  soil for FL. It was observed that  −1 fabrum were significantly different from control at 2 and 5 mg Cd kg  soil for FL. It was observed that  −1 non-significant at 5 mg Cd kg soil over control for FL (Figure 4). Main e ects of PGPRs and RNPKF  showed  a  positive  significantly  better  response  at  2  mg  Cd  kg   soil  but  remained  non‐ −1 RNPKF  showed  a  positive  significantly  better  response  at  2  mg  Cd  kg   soil  but  remained  non‐ −1 RNPKF were significant, but their interaction remained non-significant for FL at 2 and 5 mg kg soil. significant at 5 mg Cd kg  soil over control for FL (Figure 4). Main effects of PGPRs and RNPKF were  −1 significant at 5 mg Cd kg  soil over control for FL (Figure 4). Main effects of PGPRs and RNPKF were  −1 Disordinal non-significant interaction was found between PGPRs and RNPKF at 2 mg Cd kg soil, but significant, but their interaction remained non‐significant for FL at 2 and 5 mg kg  soil. Disordinal  −1 significant, but their interaction remained non‐significant for FL at 2 and 5 mg kg  soil. Disordinal  −1 the interaction was non-significant ordinal at 5 mg Cd kg soil for FL. Cadmium showed significant non‐significant  interaction  was  found  between  PGPRs  and  RNPKF  at  2  mg  Cd  kg −1   soil,  but  the  non‐significant  interaction  was  found  between  PGPRs  and  RNPKF  at  2  mg  Cd  kg   soil,  but  the  −1 but negative correlation (0.6399; p = 0.0001) with FL. Inoculation of PGPRs (0.2239; p = 0.1893) gave interaction was non‐significant ordinal at 5 mg Cd kg−1  soil for FL. Cadmium showed significant but  interaction was non‐significant ordinal at 5 mg Cd kg  soil for FL. Cadmium showed significant but  non-significant positive correlation, while RNPKF (0.3835; p = 0.021) showed positive significant negative correlation (−0.6399; p = 0.0001) with FL. Inoculation of PGPRs (0.2239; p = 0.1893) gave non‐ negative correlation (−0.6399; p = 0.0001) with FL. Inoculation of PGPRs (0.2239; p = 0.1893) gave non‐ Environments 2020, 7, x; doi: FOR PEER REVIEW  6 of 16  Environments 2020, 7, x; doi: FOR PEER REVIEW  6 of 16  Environments 2020, 7, 54 6 of 16 significant  positive  correlation,  while  RNPKF  (0.3835;  p  =  0.021)  showed  positive  significant  correlation with FL (Figure 5). The maximum increase of 19 and 29% in FL was observed from control  significant  positive  correlation,  while  RNPKF  (0.3835;  p  =  0.021)  showed  positive  significant  −1 corr where elation  RNPK with F +FL A.(Figur  fabrum e 5was ). The  applied maximum  at 2 and incr 5ease  mg of Cd 19 kg and soil, 29% rein spectively. FL was obse   rved from control correlation with FL (Figure 5). The maximum increase of 19 and 29% in FL was observed from control  −1 where RNPKF + A. fabrum was applied at 2 and 5 mg Cd kg soil, respectively. where RNPKF + A. fabrum was applied at 2 and 5 mg Cd kg  soil, respectively.  2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil 2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil ab abc abcd a abcd abcd ab abc bcd cd de de de abcd abcd abcd bcd cd de 6 de de Control RNPKF S. A. fabrum RNPKF + S. RNPKF + A. maltophilia maltophilia fabrum Control RNPKF S. A. fabrum RNPKF + S. RNPKF + A. Treatments maltophilia maltophilia fabrum Treatments Figure 4. Bitter gourd fruit length (cm) treated with PGPRs, RNPKF, and their combination under 2  −1 Figure 4. Bitter gourd fruit length (cm) treated with PGPRs, RNPKF, and their combination under 2 and 5 mg Cd kg  soil. Different small letters express significant differences at p ≤ 0.05.  Figure 4. Bitter gourd fruit length (cm) treated with PGPRs, RNPKF, and their combination under 2  and 5 mg Cd kg −1 soil. Di erent small letters express significant di erences at p  0.05. and 5 mg Cd kg  soil. Different small letters express significant differences at p ≤ 0.05.  Fruit Length Fruit Length 0.60 0.60 0.3835* 0.40 0.3835* 0.2239ns 0.40 0.20 0.2239ns 0.021 0.0001 0.1893 0.20 0.00 0.021 0.0001 0.1893 Cd PGPR RNPKF 0.00 -0.20 Cd PGPR RNPKF -0.20 -0.40 -0.40 -0.60 -0.6399* -0.60 -0.80 -0.6399* -0.80 Figure 5. Pearson correlation of Cd, PGPRs, and RNPKF for fruit length (FL). * = significant (p  0.05); Figure 5. Pearson correlation of Cd, PGPRs, and RNPKF for fruit length (FL). * = significant (p ≤ 0.05);  ns = non-significant. ns = non‐significant.  Figure 5. Pearson correlation of Cd, PGPRs, and RNPKF for fruit length (FL). * = significant (p ≤ 0.05);  3.3. Bitter Gourd Yield per Plant ns = non‐significant.  3.3. Bitter Gourd Yield per Plant  PGPRs S. maltophilia and A. fabrum and RNPKF under 2 and 5 mg Cd kg soil significantly 3.3. Bitter Gourd Yield per Plant  −1 PGPRs S. maltophilia and A. fabrum and RNPKF under 2 and 5 mg Cd kg  soil significantly (p ≤  (p  0.05) a ect bitter gourd yield per plant (YP). At 2 mg Cd kg soil, inoculation of PGPRs has −1 −1 0.05) affect bitter gourd yield per plant (YP). At 2 mg Cd kg  soil, inoculation of PGPRs has significant  significant PGPRs main  S. mal and toph interactive ilia and A. e  fabrum ects on and YP. RN Application PKF unde of r 2 RNPKF  and 5 mg has Cd a significant  kg  soil simain gnificeant ect ly on (p ≤  −1 main and interactive effects on YP. Application of RNPKF has a significant main effect on YP at 5 mg  0.05) affect bitter gourd yield per plant (YP). At 2 mg Cd kg  soil, inoculation of PGPRs has significant  YP at 5 mg Cd kg soil. Treatment RNPKF + A. fabrum was significantly di erent as compared to −1 Cd kg  soil. Treatment RNPKF + A. fabrum was significantly different as compared to control at 2 and  contr main ol and at 2 int and eract 5 mg iveCd  effe kg cts on soil YP. Cd Applica for YP t(Figur ion ofe RNP 6). ItKwas F has observed  a signific that ant the main interaction  effect on YP of PGPRs  at 5 mg  −1 −1 5 mg Cd kg  soil Cd for YP (Figure 6). It was observed that the interaction of PGPRs and RNPKF was  Cd kg  soil. Treatment RNPKF + A. fabrum was significantly different as compared to control at 2 and  and RNPKF was significant ordinal at 2 mg Cd kg soil (Figure 7A) but non-significant ordinal at −1 −1 1−1 significant ordinal at 2 mg Cd kg  soil (Figure 7A) but non‐significant ordinal at 5 mg Cd kg  soil for  55 mg  mg Cd Cd kg kg soil  soilfor  CdYP  for(Figur  YP (Feigur 7B).e Heavy 6). It was metal  observed Cd showed  that the significant  interaction negative  of PGP corr Rs  elation and RN (PK 0.4385; F was  −1 −1 YP (Figure 7B). Heavy metal Cd showed significant negative correlation (−0.4385; p = 0.0075) with YP.  significant ordinal at 2 mg Cd kg  soil (Figure 7A) but non‐significant ordinal at 5 mg Cd kg  soil for  p = 0.0075) with YP. However, PGPRs (0.5035; p = 0.0017) and RNPKF (0.3829; p = 0.0212) showed YP (Figure 7B). Heavy metal Cd showed significant negative correlation (−0.4385; p = 0.0075) with YP.  Bittergourd Fruit Length (cm) Bittergourd Fruit Length (cm) Environments 2020, 7, x; doi: FOR PEER REVIEW  7 of 16  Environments 2020, 7, x; doi: FOR PEER REVIEW  7 of 16  Environments 2020, 7, 54 7 of 16 However, However, PG  PGPRs PRs ( 0(.05.03 503 5;5; p  p=  =0. 0. 0000 171)7 )and  and RNPKF  RNPKF (0 (0 .3829 .3829 ; p;  =p  0. = 02 0.02 121) 2showe ) showe d positive d positive  signif  signif icant icant     positive correlation correlation significant  wi withth YP  YP corr  (F (F igu elation igu rer e8) 8) .with  The . The YP ma ma (Figur ximum ximum e 8incr ). incr The eaea se maximum s of e of 26 26 .5 .5 and  incr and 21 ease  21 .1% .1%  of in  26.5 in YP YP  was and  was  observed 21.1%  observed in YP  from  was from     −1 −1 observed control, control, wher  wher frome e contr RNPKF  RNPKF ol, wher +  +A. A. fabrum e fabrum RNPKF  wa wa s + asA. pplied applied fabrum at at was 2  an 2 an d applied  d 5  mg 5 mg Cd at Cd 2kg and  kg soil, 5 soil, mg  respect  Cd respect kgively ively soil, .  . respectively. 2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil 2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil 0.9 0.9 0.8 b 0.8 b bc bcd bc bcd cd cd cd cde cd cde 0.7 cde cde cde 0.7 cde cde de cde de 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0.0 0.0 Control RNPKF S. A. fabrum RNPKF + S. RNPKF + Control RNPKF S. A. fabrum RNPKF + S. RNPKF + maltophilia maltophilia A. fabrum maltophilia maltophilia A. fabrum Treatments Treatments Figure 6. Bitter gourd yield (kg) per plant treated with PGPRs, RNPKF, and their combination under  Figure 6. Bitter gourd yield (kg) per plant treated with PGPRs, RNPKF, and their combination under 2 Figure 6. Bitter gourd yield (kg) per plant treated with PGPRs, RNPKF, and their combination under  −1 2 and 5 mg Cd kg  soil. 1  Different small letters express significant differences at p ≤ 0.05.  and 5 mg Cd kg−1 soil. Di erent small letters express significant di erences at p  0.05. 2 and 5 mg Cd kg  soil. Different small letters express significant differences at p ≤ 0.05.  −1 −1 Estimated Marginal Means of YP 2 mg Cd kg  soil  Estimated Marginal Means of YP 5 mg Cd kg  soil  −1 −1 Estimated Marginal Means of YP 2 mg Cd kg  soil  Estimated Marginal Means of YP 5 mg Cd kg  soil        (A)  (B)  (A)  (B)  p < 0.05  p > 0.05  p < 0.05  p > 0.05  Figur Figure e 7. Interaction 7. Interaction  graph graph  of S. maltoph of S. maltophilia ilia (NS1), (NS1),  A. fabrA. umfabrum  (NS2), (NS2),  and RNPKF and RNPKF  at 2 (A at) and 2 (A 5 ) mg and Cd  Figur −1 e 7. Interaction graph of S. maltophilia (NS1), A. fabrum (NS2), and RNPKF at 2 (A) and 5 mg Cd  kg  so 5 mg il (B Cd ) for kg bitter soil go (B)ur ford bitter yield gour per d pla yield nt (YP per).plant   (YP). −1 kg  soil (B) for bitter gourd yield per plant (YP).  Yield per Plant (kg / plant) Yield per Plant (kg / plant) Environments 2020, 7, x; doi: FOR PEER REVIEW  8 of 16  Environments 2020, 7, 54 8 of 16 Environments 2020, 7, x; doi: FOR PEER REVIEW  8 of 16  Yield per Plant Yield per Plant 0.60 0.5035* 0.60 0.5035* 0.3829* 0.40 0.3829* 0.40 0.20 0.20 0.0075 0.0212 0.0017 0.0075 0.0212 0.00 0.0017 0.00 Cd PGPR RNPKF Cd PGPR RNPKF -0.20 -0.20 -0.40 -0.40 ̶ 0.4385* ̶ 0.4385* -0.60 -0.60 Figure 8. Pearson correlation of Cd, PGPRs, and RNPKF for yield per plant (YP)* = significant (p ≤  Figure 8. Pearson correlation of Cd, PGPRs, and RNPKF for yield per plant (YP)* = significant (p  0.05); Figure 8. Pearson correlation of Cd, PGPRs, and RNPKF for yield per plant (YP)* = significant (p ≤  0.05); ns = non‐significant.  ns = non-significant. 0.05); ns = non‐significant.  3.4. Nitrogen Concentration in Bitter Gourd 3.4. Nitrogen Concentration in Bitter Gourd   3.4. Nitrogen Concentration in Bitter Gourd   PGPRs and RNPKF significantly (p  0.05) changed the nitrogen concentration of bitter gourd PGPRs and RNPKF significantly (p ≤ 0.05) changed the nitrogen concentration of bitter gourd  (NB) under di erent levels of Cd. Main e ects of PGPRs and RNPKF were significant on NB at 2 (NB)PGPRs  under  d and iffer RNPKF ent level signif s of Cd. ica nMai tly n( pe ≤ ffect  0.05 s of )  ch PGP ange Rsd and  the RN  nitrogen PKF were  concentration  significant  on of  NB bitter  at  2go and urd    −1 and 5 mg kg soil. However, the interaction of PGPRs and RNPKF was non-significant, ordinal at 2 (NB 5 mg ) und  kg e rsoil.  differ  However, ent level the s of  int Cd. eract  Maiion n e of ffect  PGP s ofR sPGP  andR RN s and PKF RN was PK Fnon  were ‐signif  sign ica ifica nt, nordin t on NB al at at 2  2and  and    −1 −1 and 5 mg Cd kg soil for NB. It was observed that RNPKF + S. maltophilia and RNPKF + A. fabrum 55 mg  mg kg  Cd  kg soil.  soil However,  for NB. the  It was  inte ract observed ion of  tha PGP t RNPK Rs andF  RN + S.PKF  maltop  was hilia non  and‐si gnif RNPica KFn t,+  ordin A. fabrum al at  were 2 and    −1 −1 wer sign e if significantly icantly different di er as ent coas mpa compar red to ed con totro contr l at 2 ol an atd 25 and mg Cd 5 mg  kg Cd soil kg for soil NB (Fi forgNB ure (Figur 9). Heavy e 9).  5 mg Cd kg  soil for NB. It was observed that RNPKF + S. maltophilia and RNPKF + A. fabrum were  −1 Heavy metal Cd showed significant negative correlation (0.4812; p = 0.0030) with NB. However, PGPRs sign meta ificant l Cd lsho y different wed signific  as coant mpa  negative red to  co conrrelation trol at 2  (an−0.48 d 512 mg ; p  Cd = 0.00  kg30) soil  with  for NB  NB.  However, (Figure 9) .PGP  Heavy Rs    (0.4391; (0.4391;p =p 0.0074 =  0.00)7showed 4)  showsignificant ed  significand ant  and RNPKF   RNPKF (0.2041;   (0.204 p =1; 0.2324)   p  =  0.232 showed 4)  sho non-significant wed  non‐signi positive ficant  metal Cd showed significant negative correlation (−0.4812; p = 0.0030) with NB. However, PGPRs  corr positive elation cor with relation NB (Figur with NB e 10 (F ).igure The 10 maximum ). The maximum increase incre of 48 ase and  of 48 56%  and in 56 NB % in was  NBobserved  was observed from  (0.4391;  p  =  0.0074)  showed  significant  and  RNPKF  (0.2041;  p  =  0.2324)  showed  non‐significant  −1 from control where RNPKF + S. maltophilia was applied at 2 and 5 mg Cd kg  soil, respectively.  control where RNPKF + S. maltophilia was applied at 2 and 5 mg Cd kg soil, respectively. positive correlation with NB (Figure 10). The maximum increase of 48 and 56% in NB was observed  −1 from control where RNPKF + S. maltophilia was applied at 2 and 5 mg Cd kg  soil, respectively.  2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil 2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil 4.5 ab 4.0 4.5 abc a abcd ab cd bcd cd 3.5 4.0 abc abcd de cd cde bcdcde cd 3.0 3.5 de 2.5 cde cde 3.0 2.0 2.5 1.5 2.0 1.0 1.5 0.5 1.0 0.0 0.5 Control RNPKF S. A. fabrum RNPKF + S. RNPKF + A. 0.0 maltophilia maltophilia fabrum Control RNPKF S. A. fabrum RNPKF + S. RNPKF + A. Figure 9. Nitrogen concentration in bitter maltophilia gourd (%) treated with PGPRs, RNPKF maltophilia , and their combination fabrum Figure  9.  Nitrogen  concentration  in  bitter  gourd  (%)  treated  with  PGPRs,  RNPKF,  and  their  under 2 and 5 mg Cd kg soil. Di erent small letters express significant di erences (p  0.05). −1 combination under 2 and 5 mg Cd kg  soil. Different small letters express significant differences (p  Figure  9.  Nitrogen  concentration  in  bitter  gourd  (%)  treated  with  PGPRs,  RNPKF,  and  their  ≤ 0.05).  −1 combination under 2 and 5 mg Cd kg  soil. Different small letters express significant differences (p  ≤ 0.05).  Nitrogen in Bittergourd (%) Nitrogen in Bittergourd (%) Environments 2020, 7, x; doi: FOR PEER REVIEW  9 of 16  Environments 2020, 7, 54 9 of 16 Environments 2020, 7, x; doi: FOR PEER REVIEW  9 of 16  Nitrogen concentration 0.60 Nitrogen concentration 0.4391* 0.60 0.40 0.4391* 0.2041ns 0.40 0.20 0.2324 0.2041ns 0.0074 0.003 0.20 0.00 0.2324 Cd PGPR RNPKF 0.0074 0.003 0.00 -0.20 Cd PGPR RNPKF -0.20 -0.40 ̶ 0.4812* -0.40 -0.60 ̶ 0.4812* -0.60 Figure 10. Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd nitrogen concentration  (NB) * = significant (p ≤ 0.05); ns = non‐significant.  Figure 10. Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd nitrogen concentration (NB) Figure 10. Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd nitrogen concentration  * = significant (p  0.05); ns = non-significant. 3.5. Phosphorus Concentration in Bitter Gourd   (NB) * = significant (p ≤ 0.05); ns = non‐significant.  3.5. Phosphorus Concentration in Bitter Gourd −1 Effect of PGPRs and RNPKF under 2 and 5 mg kg  soil was significant (p ≤ 0.05) on phosphorus  3.5. Phosphorus Concentration in Bitter Gourd   E ect of PGPRs and RNPKF under 2 and 5 mg kg soil was significant (p  0.05) on phosphorus concentration of bitter gourd (PB). Treatments RNPKF, RNPKF + S. maltophilia, RNPKF + A. fabrum,  −1 −1 concentration and RNPKF differed of bitter signific gourdantly (PB). at Tr 5 eatments  mg Cd kg RNPKF  soil over , RNPKF  contr+olS. for maltophilia  PB (Figur ,e RNPKF  11). App +lication A. fabrum,  of  Effect of PGPRs and RNPKF under 2 and 5 mg kg  soil was significant (p ≤ 0.05) on phosphorus  −1 −1 and RNPKF di ered significantly at 5 mg Cd kg soil over control for PB (Figure 11). Application of concentration RNPKF and PGPRs  of bitter  show  gourd ed a  (PB sign).if Treat icantm ma ents in  eRN ffect PK on F,  PB RNPKF  at 2 mg +  S. Cd maltop  kg  soil. hilia At , RN  5 mg PKF Cd +  A. kg fa bsoil, rum ,  1 1 −1 RNPKF and PGPRs  RNPK  and and FPGPRs   differed RNPKF showed   signific have aa antly signific significant  atant  5 mg  ma main  Cd in  ekg and ect  soi in on tel ract over PB at ive contr 2 ef mg fec oCd lt  for on kg  PB PB. (Figu soil.  Ordinal At re 11 5  mg int ). App eCd ractlkg ication ion wa soil,  sof    −1 −1 −1 PGPRs found and between RNPKF   PGP have Rs  aan significant d  RNPKFmain   at  2 and mg  interactive Cd  kg   soeil   but ect on signi PB.fica Orn dinal t  ord interaction inal  interact was ionfound   was  RNPKF and PGPRs showed a significant main effect on PB at 2 mg Cd kg  soil. At 5 mg Cd kg  soil,  −1 observed  at  5  mg  Cd  kg   soil  (Figure  12A,B)  for  PB.  Cadmium  showed  a  significant  negative  between PGPRs and PGPRs  RNPKF and RNPKF have a  at signific 2 mgant Cd ma kg in soil andbut  inte significant ractive effec ordinal t on PB. interaction  Ordinalwas  inteobserved raction wa ats  −1 5correlation mg Cd kg (−0. soil 661 (Figur 4; p = e0.00 12A,B) 01) with for PB. BDP. Cadmium  However, showed  PGPR (a0.253 significant 7; p = 0.19 negative 53) showed corr elation non‐signi (0.6614; ficant  found  between  PGPRs  and  RNPKF  at  2  mg  Cd  kg   soil  but  significant  ordinal  interaction  was  −1 and  RNPKF  (0.4422;  p  =  0.0069)  showed  significant  positive  correlation  with  PB  (Figure  13).  The  pobserved = 0.0001)  at with   5  mg BDP  Cd . However   kg   soil , PGPR   (Figure (0.2537;   12A,B) p =  0.1953) for  PB.showed   Cadmiu non-significant m  showed  a  signific and RNPKF ant  negativ (0.4422;e  maximum increase of 29.5% in PB was observed from control where RNPKF + A. fabrum was applied  p = 0.0069) showed significant positive correlation with PB (Figure 13). The maximum increase of correlation (−0.6614; p = 0.0001) with BDP. However, PGPR (0.2537; p = 0.1953) showed non‐significant  −1 1 at 2 mg Cd kg  soil.  29.5% and  RN inPKF PB was   (0.442 observed 2;  p  =  0.00 from69) contr   showe ol wher d  signific e RNPKF ant  positive + A. fabrum   corr was elation applied   with at  PB 2 mg   (Fig Cd ure kg  13).soil.   The  maximum increase of 29.5% in PB was observed from control where RNPKF + A. fabrum was applied  −1 at 2 mg Cd kg  soil.  2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil 1.0 2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil ab a 0.8 1.0 abc bcd bcd cd cd cd cd ab a de 0.6 0.8 abc bcd bcd cd cd cd cd de 0.4 0.6 0.2 0.4 0.0 0.2 Control RNPKF S. A. fabrum RNPKF + S. RNPKF + maltophilia maltophilia A. fabrum 0.0 Figure 11. Phosphorus concentration in bitter gourd (%) treated with PGPRs, RNPKF, and their Control RNPKF S. A. fabrum RNPKF + S. RNPKF + Figure  11.  Phosphorus  concentration  in  bitter  gourd  (%)  treated  with  PGPRs,  RNPKF,  and  their  combination under 2 and 5 mg Cd kg soil. Di erent small letters express significant di erences −1 maltophilia maltophilia A. fabrum combination under 2 and 5 mg Cd kg  soil. Different small letters express significant differences (p  (p  0.05). ≤ 0.05).  Figure  11.  Phosphorus  concentration  in  bitter  gourd  (%)  treated  with  PGPRs,  RNPKF,  and  their  −1 combination under 2 and 5 mg Cd kg  soil. Different small letters express significant differences (p  ≤ 0.05).  Phosphorus in Bittergourd Phosphorus in Bittergourd -1 -1 (mg kg ) (mg kg ) Environments 2020, 7, x; doi: FOR PEER REVIEW  10 of 16  Environments 2020, 7, 54 10 of 16 Environments 2020, 7, x; doi: FOR PEER REVIEW  10 of 16  −1 −1 Estimated Marginal Means of PB 2 mg Cd kg  soil  Estimated Marginal Means of PB 5 mg Cd kg  soil  −1 −1 Estimated Marginal Means of PB 2 mg Cd kg  soil  Estimated Marginal Means of PB 5 mg Cd kg  soil     (A)  (B)     p > 0.05  p < 0.05  (A)  (B)  p > 0.05  p < 0.05  Figure 12. Interaction graphs of S. maltophilia (NS1), A. fabrum (NS2), and RNPKF at 2 (A) and 5 mg  −1 Cd kg  soil (B) for phosphorus concentration in bitter gourd (PB).  Figure 12. Interaction graphs of S. maltophilia (NS1), A. fabrum (NS2), and RNPKF at 2 (A) and 5 mg  Figure 12. Interaction graphs of S. maltophilia (NS1), A. fabrum (NS2), and RNPKF at 2 (A) and −1 5 mg Cd kg soil (B) for phosphorus concentration in bitter gourd (PB). Cd kg  soil (B) for phosphorus concentration in bitter gourd (PB).  Phosphorus concentration Phosphorus concentration 0.60 0.4422* 0.60 0.40 0.4422* 0.1953ns 0.40 0.1953ns 0.20 0.0069 0.2537 0.0001 0.20 0.00 0.0069 0.2537 0.0001 Cd PGPR RNPKF 0.00 -0.20 Cd PGPR RNPKF -0.20 -0.40 -0.40 -0.60 -0.60 ̶ 0.6614* -0.80 ̶ 0.6614* -0.80 Figure 13. Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd phosphorus concentration Figure 13. Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd phosphorus concentration  (NB). * = significant (p  0.05); ns = non-significant. (NB). * = significant (p ≤ 0.05); ns = non‐significant.  Figure 13. Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd phosphorus concentration  3.6. Potassium Concentration in Bitter Gourd (NB). * = significant (p ≤ 0.05); ns = non‐significant.  3.6. Potassium Concentration in Bitter Gourd   Influence of PGPRs and RNPKF 2 and 5 mg kg soil was significant (p  0.05) on potassium 3.6. Potassium Concentration in Bitter Gourd   concentration of bitter gourd (KB). It was also observed −1 that RNPKF + S. maltophilia and RNPKF + Influence of PGPRs and RNPKF 2 and 5 mg kg  soil was significant (p ≤ 0.05) on potassium  A. fabrum di ered significantly for KB at 5 mg Cd kg soil (Figure 14). Both PGPRs and RNPKF have −1 concentration of bitter gourd (KB). It was also observed that RNPKF + S. maltophilia and RNPKF + A.  Influence of PGPRs and RNPKF 2 and 5 mg kg  soil was significant (p ≤ 0.05) on potassium  −1 a significant main e ect on KB at 2 and 5 mg Cd kg soil. Disordinal non-significant interaction was fabrum differed significantly for KB at 5 mg Cd kg  soil (Figure 14). Both PGPRs and RNPKF have a  concentration of bitter gourd (KB). It was also observed that RNPKF + S. maltophilia and RNPKF + A.  found between PGPRs and RNPKF at 2 mg Cd kg−1 soil and but ordinal interaction was observed significant main effect on KB at 2 and 5 mg Cd kg −1  soil. Disordinal non‐significant interaction was  fabrum differed significantly for KB at 5 mg Cd kg  soil (Figure 14). Both PGPRs and RNPKF have a  −1 at 5 mg Cd kg soil for KB. Di erent levels of Cd showed significant negative correlation (0.4904; found between PGPRs and RNPKF at 2 mg Cd kg−1 soil and but ordinal interaction was observed at  significant main effect on KB at 2 and 5 mg Cd kg  soil. Disordinal non‐significant interaction was  p = 0.0024) with −1 KB. However, PGPR (0.5516; p = 0.0005) and RNPKF (0.3840; p = 0.0208) showed 5 mg Cd kg  soil for KB. Different levels of Cd showe −1 d significant negative correlation (−0.4904; p =  found between PGPRs and RNPKF at 2 mg Cd kg  soil and but ordinal interaction was observed at  significant positive correlation with KB (Figure 15). Application of RNPKF + S. maltophilia, RNPKF 0.0024)  with −1  KB.  However,  PGPR  (0.5516;  p  =  0.0005)  and  RNPKF  (0.3840;  p  =  0.0208)  showed  5 mg Cd kg  soil for KB. Different levels of Cd showed significant negative correlation (−0.4904; p =  + A. fabrum, RNPKF, S. maltophilia and A. fabrum were significantly di erent as compared to control significant positive correlation with KB (Figure 15). Application of RNPKF + S. maltophilia, RNPKF +  0.0024)  with  KB.  However,  PGPR  (0.5516;  p  =  0.0005)  and  RNPKF  (0.3840;  p  =  0.0208)  showed  at 2 mg Cd kg soil for KB. The maximum increase of 72 and 55% in KB was observed from control A. fabrum, RNPKF, S. maltophilia and A. fabrum were significantly different as compared to control at  significant positive correlation with KB (Figure 15). Application of RNPKF + S. maltophilia, RNPKF +  where RNPKF + A. fabrum was applied at 2 and 5 mg Cd kg soil, respectively. A. fabrum, RNPKF, S. maltophilia and A. fabrum were significantly different as compared to control at  Environments 2020, 7, x; doi: FOR PEER REVIEW  11 of 16  −1 2 mg Cd kg  soil for KB. The maximum increase of 72 and 55% in KB was observed from control  −1 where RNPKF + A. fabrum was applied at 2 and 5 mg Cd kg  soil, respectively.  Environments 2020, 7, x; doi: FOR PEER REVIEW  11 of 16  Environments 2020, 7, 54 2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil 11 of 16 −1 2 mg Cd kg  soil for KB. The maximum increase of 72 and 55% in KB was observed from control  −1 9.0 where RNPKF + A. fabrum was applied at 2 and 5 mg Cd kg  soil, respectively.  ab 2.0 mg Cd kg-1 Soil 5.0 mg Cd kg-1 Soil 7.5 bc bcd bcd bcde cde 9.0 6.0 cdef def ef ab 7.5 bc bcd 4.5 bcd bcde cde 6.0 cdef def ef 3.0 4.5 1.5 3.0 0.0 1.5 Control RNPKF S. maltophilia A. fabrum RNPKF + S. RNPKF + A. maltophilia fabrum 0.0 Control RNPKF S. maltophilia A. fabrum RNPKF + S. RNPKF + A.   maltophilia fabrum Figure  14.  Potassium  concentration  in  bitter  gourd  (%)  treated  with  PGPRs,  RNPKF,  and  their  −1   combinat Figureion 14. under Potassium  2 and concentration 5 mg Cd kg in soil. bitter  Different gourd  (%) small treated  letters with  express PGPRs,  significant RNPKF, and differen theirces (p  combination under 2 and 5 mg Cd kg soil. Di erent small letters express significant di erences ≤ 0.05).  Figure  14.  Potassium  concentration  in  bitter  gourd  (%)  treated  with  PGPRs,  RNPKF,  and  their  (p  0.05). −1 combination under 2 and 5 mg Cd kg  soil. Different small letters express significant differences (p  ≤ 0.05).  Potassium concentration 0.80 Potassium concentration 0.5516* 0.80 0.60 0.384* 0.5516* 0.60 0.40 0.384* 0.40 0.20 0.0208 0.0024 0.0005 0.20 0.00 0.0208 0.0024 0.0005 Cd PGPR RNPKF 0.00 -0.20 Cd PGPR RNPKF -0.20 -0.40 -0.40 ̶ 0.4904* -0.60 ̶ 0.4904* -0.60 Figure 15. Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd potassium concentration Figure 15. Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd potassium concentration  (NB). * = significant (p  0.05); ns = non-significant. Figure 15. Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd potassium concentration  (NB). * = significant (p ≤ 0.05); ns = non‐significant.  4. Discussion (NB). * = significant (p ≤ 0.05); ns = non‐significant.  4. Discussion  A significant decrease in fruit length, fresh weight, and yield per plant of bitter gourd were 4. Discussion  observed in control at 5 mg Cd kg soil. Low uptake of N, P, and K in bitter gourd under Cd toxicity A significant decrease in fruit length, fresh weight, and yield per plant of bitter gourd were  A significant decrease in fruit length, fresh weight, and yield per plant of bitter gourd were  −1 might be a major factor for reduction in yield, fruit length, and fresh weight. Higher biosynthesis observed in control at 5 mg Cd kg  soil. Low uptake of N, P, and K in bitter gourd under Cd toxicity  −1 observed in control at 5 mg Cd kg  soil. Low uptake of N, P, and K in bitter gourd under Cd toxicity  of stress ethylene might be an allied factor responsible for a significant decline in yield of bitter might be a major factor for reduction in yield, fruit length, and fresh weight. Higher biosynthesis of  might be a major factor for reduction in yield, fruit length, and fresh weight. Higher biosynthesis of  gourd under Cd stress. According to Sanita di Toppi and Gabbrielli [7], accumulation of Cd beyond stress ethylene might be an allied factor responsible for a significant decline in yield of bitter gourd  stress ethylene might be an allied factor responsible for a significant decline in yield of bitter gourd  safe limit disturbed the nutrients homeostasis which played an imperative role in reduction of root under Cd stress. According to Sanita di Toppi and Gabbrielli [7], accumulation of Cd beyond safe  under Cd stress. According to Sanita di Toppi and Gabbrielli [7], accumulation of Cd beyond safe  and shoot elongation. Cadmium in plants also competes with divalent nutrients ions and decreases limit disturbed the nutrients homeostasis which played an imperative role in reduction of root and  limit disturbed the nutrients homeostasis which played an imperative role in reduction of root and  their uptake in plants [16]. Under Cd toxicity, transmembrane carriers in roots become unable to shoot elongation. Cadmium in plants also competes with divalent nutrients ions and decreases their  shoot elongation. Cadmium in plants also competes with divalent nutrients ions and decreases their  distinguish between non-essential Cd and essential divalent nutrients during their uptake [53,54]. uptake  in  plants  [16].  Under  Cd  toxicity,  transmembrane  carriers  in  roots  become  unable  to  uptake  in  plants  [16].  Under  Cd  toxicity,  transmembrane  carriers  in  roots  become  unable  to  Glick et al. [55] also documented that biosynthesis of endogenous stress ethylene under abiotic stress conditions, negatively a ects the productivity of the crop. Toxicity of heavy metals causes abnormal division of cell thus induced chromosomal aberration in plants [56]. This resulted in a decrease of protochlorophyllide reductase activity. Such disturbance in plants also induced chlorosis in leaves [57]. -1 -1 Potassium in Bittergourd (mg kg ) Potassium in Bittergourd (mg kg ) Environments 2020, 7, 54 12 of 16 Furthermore, Matile et al. [58] suggested the decomposition of lipids in cell wall when ethylene concentration is increased. They argued that ethylene when contact with chlorophyllase (chlase) gene it degrades chlorophyll caused in chlorosis. Furthermore, application of RNPKF + A. fabrum di ered significantly better from the sole application of control for improvement in N, P and K. The improvement in N, P, and K mitigate the adverse impacts of Cd in bitter gourd. Pankovic ´ et al. [59] observed that improvement in N uptake of sunflower alleviants the inhibitory influences of Cd [22,23,27,28,32]. Higher N facilitates in activity of Rubisco by an increase in soluble protein contents. Application of N in NH form is ecacious in decreasing the Cd uptake due to antagonistic relationship [60]. Findings of the current experiment also support the above argument. Better N in bitter gourd was observed where yield was improved over control even under Cd toxicity. Under Cd stress, plants start producing N metabolites, i.e., proline that causes phytochelation and decreases the intake of Cd [61]. Application of phosphorus neutralizes the adverse impacts of Cd and improve the yield of crops [62]. Improvement of P uptake in plants enhances the synthesis of glutathione that prevents membrane damages caused by Cd [63]. Balance K concentration decreases the generation of reactive oxidative species (ROS) and inhibits the NADPH oxidase [64]. Moreover, less generation of stress ethylene by inoculation of A. fabrum and RNPKF + A. fabrum might be another major factor responsible for the enhancement in bitter gourd growth and yield in the current study. Both PGPRs were capable to produce ACC deaminase, which cleaves ethylene into intermediate compounds. Similar kinds of results were also documented by many scientists [25,26,30,31]. Glick et al. [44] proposed that enzyme ACC deaminase break ethylene into -ketobutyrate and ammonia [65,66]. Accumulated ethylene in roots moved towards rhizosphere; thus, ethylene becomes low in plant roots, and stress is alleviated. Similarly, Tripathi et al. [67] reported growth hormones, indole acetic acid, improved the root elongation for better uptake of nutrients [24]. 5. Conclusions It is concluded that PGPR, A. fabrum has more potential over S. maltophilia to alleviate Cd induced stress in bitter gourd. Inoculation of A. fabrum with RNPKF is an ecacious approach to improve N, P, and K concentration in bitter gourd. The combined use of RNPKF and A. fabrum can increase the number of bitter gourds per plant, bitter gourd fruit length, and yield per plant by alleviating 5 mg Cd kg soil induced toxicity. 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Environments – Multidisciplinary Digital Publishing Institute

Published: Jul 26, 2020

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