Alleviation of Cadmium Adverse E ﬀ ects by Improving Nutrients Uptake in Bitter Gourd through Cadmium Tolerant Rhizobacteria

: 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 ﬀ 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 ﬀ 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 ﬀ erent concentration of soil cadmium (2 and 5 mg kg − 1 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 ﬀ erent concentrations of soil cadmium (2 and 5 mg kg − 1 soil), respectively. In conclusion, we suggest that A. fabrum with RNPKF can more e ﬃ caciously enhance N, K, and yield of bitter gourd under cadmium toxicity. to ectiveness of in the of uptake; of on


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][4][5][6]. Among different 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 −1 soil is considered a safe limit, but depending upon parent material, it can be accumulated up to 3.0 mg kg −1 soil [8]. Being a part of phosphate fertilizers (up to 4.4 mg kg −1 ), it is easily taken up by crops as Cd-supplement [9,10].
In addition, ethylene (C 2 H 4 ) is a plant-signaling molecule. It is involved in seed germination 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 affects gas exchange attributes, nutrients and water uptake, and yield of different crops under any stress conditions [30,31].
Among different 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 affected 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 efficacy of ACC deaminase producing PGPR with recommended NPK fertilizers (RNPKF) on bitter gourd nutrients uptake and yield under Cd toxicity. The present study aimed to explore (1) effectiveness of rhizobacteria in the improvement of nutrients uptake; (2) effect 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 effects of Cd in bitter gourd for yield improvement.

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 −1 soil. Artificial toxicity of Cd was developed by using analytical grade salt of CdCl 2 [25]. As per treatment plan, two levels of Cd were maintained, i.e., 2 and 5 ppm (mg kg −1 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.

Collection of Bacterial Strains and Broth
ACC deaminase producing PGPRs S. maltophilia (ACC deaminase activity = 71.78 µmol α-ketobutyrate mg −1 protein h −1 ) and A. fabrum (ACC deaminase activity = 432.6 µmol α-ketobutyrate mg −1 protein h −1 ) 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 −1 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 achieve 0.45 nm OD, to achieve a uniform population of 10 7 -10 8 colony forming units (CFU) mL -1 .

Seeds Sterilization and Sowing
HgCl 2 (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.

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 −1 ) urea, K 2 HPO 4 and K 2 SO 4 were applied.

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]. 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 2 SO 4 , and digestion mixture (K 2 SO 4 (100):CuSO 4 .5H 2 O (10):FeSO 4 (1)). Distillation was performed in Kjeldahl distillation apparatus, using boric acid as a collector [52].

Statistical Analyses
One-way ANOVA was used to assess the effects of treatments. Two factorial ANOVA was conducted separately to compare PGPRs and RNPK interaction under different levels of Cd. Treatment comparison was computed at p ≤ 0.05 by Tukey's Test.

Number of Bitter Gourds per Plant
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 interactive effects on BDP at 2 and 5 mg kg −1 soil. Application of RNPKF + S. maltophilia, RNPKF + A. fabrum, RNPKF, S. maltophilia and A. fabrum showed significant positive effect over control at 2 and 5 mg Cd kg −1 soil for BDP ( Figure 1). Interaction between PGPRs and RNPKF at 2 mg Cd kg −1 soil ( Figure 2A) and 5 mg Cd kg −1 soil ( Figure 2B) were significant ordinal for BDP ( Figure 2B). It was noted that Cd showed non-significant negative correlation (−0.1451; p = 0.3986) with BDP. However, PGPR (0.5863; p = 0.0002) and RNPKF (0.3237; p = 0.0541) showed positive significant correlation with BDP ( Figure 3). The maximum increase of 34% and 68% in BDP was observed from control where RNPKF + A. fabrum was applied at 2 and 5 mg Cd kg −1 soil, respectively.

Statistical Analyses
One-way ANOVA was used to assess the effects of treatments. Two factorial ANOVA was conducted separately to compare PGPRs and RNPK interaction under different levels of Cd. Treatment comparison was computed at p ≤ 0.05 by Tukey's Test.

Number of Bitter Gourds per Plant
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 interactive effects on BDP at 2 and 5 mg kg −1 soil. Application of RNPKF + S. maltophilia, RNPKF + A. fabrum, RNPKF, S. maltophilia and A. fabrum showed significant positive effect over control at 2 and 5 mg Cd kg −1 soil for BDP ( Figure 1). Interaction between PGPRs and RNPKF at 2 mg Cd kg −1 soil ( Figure 2A) and 5 mg Cd kg −1 soil ( Figure 2B) were significant ordinal for BDP ( Figure 2B). It was noted that Cd showed non-significant negative correlation (−0.1451; p = 0.3986) with BDP. However, PGPR (0.5863; p = 0.0002) and RNPKF (0.3237; p = 0.0541) showed positive significant correlation with BDP ( Figure 3). The maximum increase of 34% and 68% in BDP was observed from control where RNPKF + A. fabrum was applied at 2 and 5 mg Cd kg −1 soil, respectively.

Bitter Gourd Fruit Length
Effects of PGPRs inoculation 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 + A. fabrum were significantly different from control at 2 and 5 mg Cd kg −1 soil for FL. It was observed that RNPKF showed a positive significantly better response at 2 mg Cd kg −1 soil but remained nonsignificant at 5 mg Cd kg −1 soil over control for FL ( Figure 4). Main effects of PGPRs and RNPKF were significant, but their interaction remained non-significant for FL at 2 and 5 mg kg −1 soil. Disordinal non-significant interaction was found between PGPRs and RNPKF at 2 mg Cd kg −1 soil, but the interaction was non-significant ordinal at 5 mg Cd kg −1 soil for FL. Cadmium showed significant but negative correlation (−0.6399; p = 0.0001) with FL. Inoculation of PGPRs (0.2239; p = 0.1893) gave non-

Bitter Gourd Fruit Length
Effects of PGPRs inoculation 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 + A. fabrum were significantly different from control at 2 and 5 mg Cd kg −1 soil for FL. It was observed that RNPKF showed a positive significantly better response at 2 mg Cd kg −1 soil but remained nonsignificant at 5 mg Cd kg −1 soil over control for FL ( Figure 4). Main effects of PGPRs and RNPKF were significant, but their interaction remained non-significant for FL at 2 and 5 mg kg −1 soil. Disordinal non-significant interaction was found between PGPRs and RNPKF at 2 mg Cd kg −1 soil, but the interaction was non-significant ordinal at 5 mg Cd kg −1 soil for FL. Cadmium showed significant but negative correlation (−0.6399; p = 0.0001) with FL. Inoculation of PGPRs (0.2239; p = 0.1893) gave non-

Bitter Gourd Fruit Length
Effects of PGPRs inoculation 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 + A. fabrum were significantly different from control at 2 and 5 mg Cd kg −1 soil for FL. It was observed that RNPKF showed a positive significantly better response at 2 mg Cd kg −1 soil but remained non-significant at 5 mg Cd kg −1 soil over control for FL ( Figure 4). Main effects of PGPRs and RNPKF were significant, but their interaction remained non-significant for FL at 2 and 5 mg kg −1 soil. Disordinal non-significant interaction was found between PGPRs and RNPKF at 2 mg Cd kg −1 soil, but the interaction was non-significant ordinal at 5 mg Cd kg −1 soil for FL. Cadmium showed significant but negative correlation (−0.6399; p = 0.0001) with FL. Inoculation of PGPRs (0.2239; p = 0.1893) gave non-significant positive correlation, while RNPKF (0.3835; p = 0.021) showed positive significant Environments 2020, 7, 54 6 of 16 correlation with FL ( Figure 5). The maximum increase of 19 and 29% in FL was observed from control where RNPKF + A. fabrum was applied at 2 and 5 mg Cd kg −1 soil, respectively.
Environments 2020, 7, x; doi: FOR PEER REVIEW 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 where RNPKF + A. fabrum was applied at 2 and 5 mg Cd kg −1 soil, respectively.

Bitter Gourd Yield per Plant
PGPRs S. maltophilia and A. fabrum and RNPKF under 2 and 5 mg Cd kg −1 soil significantly (p ≤ 0.05) affect bitter gourd yield per plant (YP). At 2 mg Cd kg −1 soil, inoculation of PGPRs has significant main and interactive effects on YP. Application of RNPKF has a significant main effect on YP at 5 mg Cd kg −1 soil. Treatment RNPKF + A. fabrum was significantly different as compared to control at 2 and 5 mg Cd kg −1 soil Cd for YP ( Figure 6). It was observed that the interaction of PGPRs and RNPKF was significant ordinal at 2 mg Cd kg −1 soil ( Figure 7A) but non-significant ordinal at 5 mg Cd kg −1 soil for YP ( Figure 7B). Heavy metal Cd showed significant negative correlation (−0.4385; p = 0.0075) with YP.

Bitter Gourd Yield per Plant
PGPRs S. maltophilia and A. fabrum and RNPKF under 2 and 5 mg Cd kg −1 soil significantly (p ≤ 0.05) affect bitter gourd yield per plant (YP). At 2 mg Cd kg −1 soil, inoculation of PGPRs has significant main and interactive effects on YP. Application of RNPKF has a significant main effect on YP at 5 mg Cd kg −1 soil. Treatment RNPKF + A. fabrum was significantly different as compared to control at 2 and 5 mg Cd kg −1 soil Cd for YP ( Figure 6). It was observed that the interaction of PGPRs and RNPKF was significant ordinal at 2 mg Cd kg −1 soil ( Figure 7A) but non-significant ordinal at 5 mg Cd kg −1 soil for YP ( Figure 7B). Heavy metal Cd showed significant negative correlation (−0.4385; p = 0.0075) with YP.

Bitter Gourd Yield per Plant
PGPRs S. maltophilia and A. fabrum and RNPKF under 2 and 5 mg Cd kg −1 soil significantly (p ≤ 0.05) affect bitter gourd yield per plant (YP). At 2 mg Cd kg −1 soil, inoculation of PGPRs has significant main and interactive effects on YP. Application of RNPKF has a significant main effect on YP at 5 mg Cd kg −1 soil. Treatment RNPKF + A. fabrum was significantly different as compared to control at 2 and 5 mg Cd kg −1 soil Cd for YP ( Figure 6). It was observed that the interaction of PGPRs and RNPKF was significant ordinal at 2 mg Cd kg −1 soil ( Figure 7A) but non-significant ordinal at 5 mg Cd kg −1 soil for YP ( Figure 7B

Nitrogen Concentration in Bitter Gourd
PGPRs and RNPKF significantly (p ≤ 0.05) changed the nitrogen concentration of bitter gourd (NB) under different levels of Cd. Main effects of PGPRs and RNPKF were significant on NB at 2 and 5 mg kg −1 soil. However, the interaction of PGPRs and RNPKF was non-significant, ordinal at 2 and 5 mg Cd kg −1 soil for NB. It was observed that RNPKF + S. maltophilia and RNPKF + A. fabrum were significantly different as compared to control at 2 and 5 mg Cd kg −1 soil for NB ( Figure 9). Heavy metal Cd showed significant negative correlation (−0.4812; p = 0.0030) with NB. However, PGPRs (0.4391; p = 0.0074) showed significant and RNPKF (0.2041; p = 0.2324) showed non-significant positive correlation with NB ( Figure 10). The maximum increase of 48 and 56% in NB was observed from control where RNPKF + S. maltophilia was applied at 2 and 5 mg Cd kg −1 soil, respectively.

Nitrogen Concentration in Bitter Gourd
PGPRs and RNPKF significantly (p ≤ 0.05) changed the nitrogen concentration of bitter gourd (NB) under different levels of Cd. Main effects of PGPRs and RNPKF were significant on NB at 2 and 5 mg kg −1 soil. However, the interaction of PGPRs and RNPKF was non-significant, ordinal at 2 and 5 mg Cd kg −1 soil for NB. It was observed that RNPKF + S. maltophilia and RNPKF + A. fabrum were significantly different as compared to control at 2 and 5 mg Cd kg −1 soil for NB ( Figure 9). Heavy metal Cd showed significant negative correlation (−0.4812; p = 0.0030) with NB. However, PGPRs (0.4391; p = 0.0074) showed significant and RNPKF (0.2041; p = 0.2324) showed non-significant positive correlation with NB ( Figure 10). The maximum increase of 48 and 56% in NB was observed from control where RNPKF + S. maltophilia was applied at 2 and 5 mg Cd kg −1 soil, respectively.

Nitrogen Concentration in Bitter Gourd
PGPRs and RNPKF significantly (p ≤ 0.05) changed the nitrogen concentration of bitter gourd (NB) under different levels of Cd. Main effects of PGPRs and RNPKF were significant on NB at 2 and 5 mg kg −1 soil. However, the interaction of PGPRs and RNPKF was non-significant, ordinal at 2 and 5 mg Cd kg −1 soil for NB. It was observed that RNPKF + S. maltophilia and RNPKF + A. fabrum were significantly different as compared to control at 2 and 5 mg Cd kg −1 soil for NB (Figure 9). Heavy metal Cd showed significant negative correlation (−0.4812; p = 0.0030) with NB. However, PGPRs (0.4391; p = 0.0074) showed significant and RNPKF (0.2041; p = 0.2324) showed non-significant positive correlation with NB ( Figure 10). The maximum increase of 48 and 56% in NB was observed from control where RNPKF + S. maltophilia was applied at 2 and 5 mg Cd kg −1 soil, respectively.

Phosphorus Concentration in Bitter Gourd
Effect of PGPRs and RNPKF under 2 and 5 mg kg −1 soil was significant (p ≤ 0.05) on phosphorus concentration of bitter gourd (PB). Treatments RNPKF, RNPKF + S. maltophilia, RNPKF + A. fabrum, and RNPKF differed significantly at 5 mg Cd kg −1 soil over control for PB ( Figure 11). Application of RNPKF and PGPRs showed a significant main effect on PB at 2 mg Cd kg −1 soil. At 5 mg Cd kg −1 soil, PGPRs and RNPKF have a significant main and interactive effect on PB. Ordinal interaction was found between PGPRs and RNPKF at 2 mg Cd kg −1 soil but significant ordinal interaction was observed at 5 mg Cd kg −1 soil ( Figure 12A

Phosphorus Concentration in Bitter Gourd
Effect of PGPRs and RNPKF under 2 and 5 mg kg −1 soil was significant (p ≤ 0.05) on phosphorus concentration of bitter gourd (PB). Treatments RNPKF, RNPKF + S. maltophilia, RNPKF + A. fabrum, and RNPKF differed significantly at 5 mg Cd kg −1 soil over control for PB ( Figure 11). Application of RNPKF and PGPRs showed a significant main effect on PB at 2 mg Cd kg −1 soil. At 5 mg Cd kg −1 soil, PGPRs and RNPKF have a significant main and interactive effect on PB. Ordinal interaction was found between PGPRs and RNPKF at 2 mg Cd kg −1 soil but significant ordinal interaction was observed at 5 mg Cd kg −1 soil ( Figure 12A,B) for PB. Cadmium showed a significant negative correlation (−0.6614; p = 0.0001) with BDP. However, PGPR (0.2537; p = 0.1953) showed non-significant and RNPKF (0.4422; p = 0.0069) showed significant positive correlation with PB ( Figure 13). The maximum increase of 29.5% in PB was observed from control where RNPKF + A. fabrum was applied at 2 mg Cd kg −1 soil.

Phosphorus Concentration in Bitter Gourd
Effect of PGPRs and RNPKF under 2 and 5 mg kg −1 soil was significant (p ≤ 0.05) on phosphorus concentration of bitter gourd (PB). Treatments RNPKF, RNPKF + S. maltophilia, RNPKF + A. fabrum, and RNPKF differed significantly at 5 mg Cd kg −1 soil over control for PB ( Figure 11). Application of RNPKF and PGPRs showed a significant main effect on PB at 2 mg Cd kg −1 soil. At 5 mg Cd kg −1 soil, PGPRs and RNPKF have a significant main and interactive effect on PB. Ordinal interaction was found between PGPRs and RNPKF at 2 mg Cd kg −1 soil but significant ordinal interaction was observed at 5 mg Cd kg −1 soil ( Figure 12A,B) for PB. Cadmium showed a significant negative correlation (−0.6614; p = 0.0001) with BDP. However, PGPR (0.2537; p = 0.1953) showed non-significant and RNPKF (0.4422; p = 0.0069) showed significant positive correlation with PB ( Figure 13). The maximum increase of 29.5% in PB was observed from control where RNPKF + A. fabrum was applied at 2 mg Cd kg −1 soil.

Potassium Concentration in Bitter Gourd
Influence of PGPRs and RNPKF 2 and 5 mg kg −1 soil was significant (p ≤ 0.05) on potassium concentration of bitter gourd (KB). It was also observed that RNPKF + S. maltophilia and RNPKF + A. fabrum differed significantly for KB at 5 mg Cd kg −1 soil (Figure 14). Both PGPRs and RNPKF have a significant main effect on KB at 2 and 5 mg Cd kg −1 soil. Disordinal non-significant interaction was found between PGPRs and RNPKF at 2 mg Cd kg −1 soil and but ordinal interaction was observed at 5 mg Cd kg −1 soil for KB. Different levels of Cd showed significant negative correlation (−0.4904; p = 0.0024) with KB. However, PGPR (0.5516; p = 0.0005) and RNPKF (0.3840; p = 0.0208) showed significant positive correlation with KB ( Figure 15). Application of RNPKF + S. maltophilia, RNPKF + A. fabrum, RNPKF, S. maltophilia and A. fabrum were significantly different as compared to control at

Potassium Concentration in Bitter Gourd
Influence of PGPRs and RNPKF 2 and 5 mg kg −1 soil was significant (p ≤ 0.05) on potassium concentration of bitter gourd (KB). It was also observed that RNPKF + S. maltophilia and RNPKF + A. fabrum differed significantly for KB at 5 mg Cd kg −1 soil ( Figure 14). Both PGPRs and RNPKF have a significant main effect on KB at 2 and 5 mg Cd kg −1 soil. Disordinal non-significant interaction was found between PGPRs and RNPKF at 2 mg Cd kg −1 soil and but ordinal interaction was observed at 5 mg Cd kg −1 soil for KB. Different levels of Cd showed significant negative correlation (−0.4904; p = 0.0024) with KB. However, PGPR (0.5516; p = 0.0005) and RNPKF (0.3840; p = 0.0208) showed significant positive correlation with KB ( Figure 15). Application of RNPKF + S. maltophilia, RNPKF + A. fabrum, RNPKF, S. maltophilia and A. fabrum were significantly different as compared to control at

Potassium Concentration in Bitter Gourd
Influence of PGPRs and RNPKF 2 and 5 mg kg −1 soil was significant (p ≤ 0.05) on potassium concentration of bitter gourd (KB). It was also observed that RNPKF + S. maltophilia and RNPKF + A. fabrum differed significantly for KB at 5 mg Cd kg −1 soil ( Figure 14). Both PGPRs and RNPKF have a significant main effect on KB at 2 and 5 mg Cd kg −1 soil. Disordinal non-significant interaction was found between PGPRs and RNPKF at 2 mg Cd kg −1 soil and but ordinal interaction was observed at 5 mg Cd kg −1 soil for KB. Different levels of Cd showed significant negative correlation (−0.4904; p = 0.0024) with KB. However, PGPR (0.5516; p = 0.0005) and RNPKF (0.3840; p = 0.0208) showed significant positive correlation with KB ( Figure 15). Application of RNPKF + S. maltophilia, RNPKF + A. fabrum, RNPKF, S. maltophilia and A. fabrum were significantly different as compared to control at 2 mg Cd kg −1 soil for KB. The maximum increase of 72 and 55% in KB was observed from control where RNPKF + A. fabrum was applied at 2 and 5 mg Cd kg −1 soil, respectively.

Discussion
A significant decrease in fruit length, fresh weight, and yield per plant of bitter gourd were observed in control at 5 mg Cd kg −1 soil. Low uptake of N, P, and K in bitter gourd under Cd toxicity might be a major factor for reduction in yield, fruit length, and fresh weight. Higher biosynthesis of stress ethylene might be an allied factor responsible for a significant decline in yield of bitter gourd under Cd stress. According to Sanita di Toppi and Gabbrielli [7], accumulation of Cd beyond safe limit disturbed the nutrients homeostasis which played an imperative role in reduction of root and shoot elongation. Cadmium in plants also competes with divalent nutrients ions and decreases their uptake in plants [16]. Under Cd toxicity, transmembrane carriers in roots become unable to Environments 2020, 7, x; doi: FOR PEER REVIEW 11 of 16 2 mg Cd kg −1 soil for KB. The maximum increase of 72 and 55% in KB was observed from control where RNPKF + A. fabrum was applied at 2 and 5 mg Cd kg −1 soil, respectively.

Discussion
A significant decrease in fruit length, fresh weight, and yield per plant of bitter gourd were observed in control at 5 mg Cd kg −1 soil. Low uptake of N, P, and K in bitter gourd under Cd toxicity might be a major factor for reduction in yield, fruit length, and fresh weight. Higher biosynthesis of stress ethylene might be an allied factor responsible for a significant decline in yield of bitter gourd under Cd stress. According to Sanita di Toppi and Gabbrielli [7], accumulation of Cd beyond safe limit disturbed the nutrients homeostasis which played an imperative role in reduction of root and shoot elongation. Cadmium in plants also competes with divalent nutrients ions and decreases their uptake in plants [16]. Under Cd toxicity, transmembrane carriers in roots become unable to

Discussion
A significant decrease in fruit length, fresh weight, and yield per plant of bitter gourd were observed in control at 5 mg Cd kg −1 soil. Low uptake of N, P, and K in bitter gourd under Cd toxicity might be a major factor for reduction in yield, fruit length, and fresh weight. Higher biosynthesis of stress ethylene might be an allied factor responsible for a significant decline in yield of bitter gourd under Cd stress. According to Sanita di Toppi and Gabbrielli [7], accumulation of Cd beyond safe limit disturbed the nutrients homeostasis which played an imperative role in reduction of root and shoot elongation. Cadmium in plants also competes with divalent nutrients ions and decreases their uptake in plants [16]. Under Cd toxicity, transmembrane carriers in roots become unable to distinguish between non-essential Cd and essential divalent nutrients during their uptake [53,54]. Glick et al. [55] also documented that biosynthesis of endogenous stress ethylene under abiotic stress conditions, negatively affects 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]. 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 differed 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. Panković 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 4 form is efficacious 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 α-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].

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 efficacious 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 −1 soil induced toxicity. However, more investigations are suggested at field level to declare A. fabrum + RNPKF as an efficacious technique to mitigate Cd stress in bitter gourd.