*3.6. Potassium Concentration in Bitter Gourd*

Influence of PGPRs and RNPKF 2 and 5 mg kg−<sup>1</sup> soil was significant (*p* <sup>≤</sup> 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−<sup>1</sup> soil (Figure 14). Both PGPRs and RNPKF have a significant main effect on KB at 2 and 5 mg Cd kg−<sup>1</sup> soil. Disordinal non-significant interaction was found between PGPRs and RNPKF at 2 mg Cd kg−<sup>1</sup> soil and but ordinal interaction was observed at 5 mg Cd kg−<sup>1</sup> 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−<sup>1</sup> 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−<sup>1</sup> soil, respectively.

**Figure 14.** Potassium concentration in bitter gourd (%) treated with PGPRs, RNPKF, and their combination under 2 and 5 mg Cd kg−<sup>1</sup> soil. Different small letters express significant differences (*p* ≤ 0.05).

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**Figure 15.** Pearson correlation of Cd, PGPRs, and RNPKF for bitter gourd potassium concentration (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 observed in control at 5 mg Cd kg−<sup>1</sup> 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. Pankovi´c 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 NH4 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].

## **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 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−<sup>1</sup> 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.

**Author Contributions:** M.Z.-u.-H. and S.D. designed and supervised the experiment and wrote the manuscript; M.N. (Muhammad Naeem) conducted research, collected data; S.D., M.B., J.H., and R.D. wrote the manuscript and conducted statistical analyses; S.F., M.A., A.A.R., Z.H.T., and M.N. (Muhammad Nasir) assisted in the preparation of manuscript and reviewed manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** This research article is part of Muhammad Naeem Thesis for the award of M.Sc. Hons. Agriculture (Soil Science) Degree.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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