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

**Muhammad Zafar-ul-Hye 1, Muhammad Naeem 1, Subhan Danish 1,\*, Shah Fahad 2,3,\*, Rahul Datta 4,\*, Mazhar Abbas 5, Ashfaq Ahmad Rahi 6, Martin Brtnicky 4,7,8, Jiˇrí Holátko 8, Zahid Hassan Tarar <sup>9</sup> and Muhammad Nasir <sup>10</sup>**


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 effects 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 effects. 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 different concentration of soil cadmium (2 and 5 mg kg−<sup>1</sup> 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 different concentrations of soil cadmium (2 and 5 mg kg−<sup>1</sup> soil), respectively. In conclusion, we suggest that *A. fabrum* with RNPKF can more efficaciously enhance N, K, and yield of bitter gourd under cadmium toxicity.

**Keywords:** ACC deaminase; heavy metal stress; PGPR; fertilizers; nutrients; yield

### **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 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−<sup>1</sup> soil is considered a safe limit, but depending upon parent material, it can be accumulated up to 3.0 mg kg−<sup>1</sup> soil [8]. Being a part of phosphate fertilizers (up to 4.4 mg kg<sup>−</sup>1), 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 affected when Cd is present in higher concentration in soil [19]. Heavy metal toxicity and physiochemical properties soil depend on the land use [20–22]. Different crops, the biological adsorption factor (mg Cd/kg plant ash, mg Cd/kg soil) based on Cd content in plant ash, is different, 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 (C2H4) 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].

To overcome these problems, inoculation of ACC deaminase producing plant growth promoting rhizobacteria (PGPR), could be an efficacious 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 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.
