**Isolation and Characterization of Plant Growth Promoting Endophytic Bacteria from Desert Plants and Their Application as Bioinoculants for Sustainable Agriculture**

**Muneera D. F. ALKahtani 1, Amr Fouda 2, Kotb A. Attia 3, Fahad Al-Otaibi 4,5, Ahmed M. Eid 2, Emad El-Din Ewais 2, Mohamed Hijri 4,6 , Marc St-Arnaud <sup>4</sup> , Saad El-Din Hassan 2, Naeem Khan <sup>7</sup> , Yaser M. Hafez <sup>8</sup> and Khaled A. A. Abdelaal 8,\***


Received: 29 July 2020; Accepted: 26 August 2020; Published: 4 September 2020

**Abstract:** Desert plants are able to survive under harsh environmental stresses inherent to arid and semiarid regions due to their association with bacterial endophytes. However, the identity, functions, and the factors that influence the association of bacterial endophytes with desert plants are poorly known. These bacterial endophytes can be used as an untapped resource to favor plant growth and development in agro-ecosystems of arid regions. The present study is therefore focused on the isolation and identification of bacterial endophytes from two native medicinal plants (*Fagonia mollis* Delile and *Achillea fragrantissima* (Forssk) Sch. Bip.) growing spontaneously in the arid region of the South Sinai (Egypt), and characterization of their plant growth promoting (PGP) traits. Thirteen putative bacterial endophytes were isolated from the leaves of both plant species and characterized for their plant growth promoting abilities using molecular and biochemical approaches, as well as greenhouse trials. Selected endophytic bacterial strains were applied to maize plants (*Zea mays* L. var. Single cross Pioneer 30K08) to further evaluate their PGP abilities under greenhouse conditions. Isolated bacterial strains have variable plant growth promoting activities. Among these activities, isolated bacterial endophytes have the efficacy of phosphate solubilizing with clear zones ranging from 7.6 ± 0.3 to 9.6 ± 0.3 mm. Additionally, the obtained bacterial endophytes increased the productivity of indole acetic acid (IAA) in broth media from 10 to 60 μg·mL−<sup>1</sup> with increasing tryptophan concentration from 1 to 5 mg·mL<sup>−</sup>1. *Bacillus* and *Brevibacillus* strains were frequently isolated from the leaves of both plant species, and had significant positive effects on plant growth and shoot phosphorus (P) and nitrogen (N) contents. Results suggest that these endophytes are good candidates as plant growth promoting inoculants to help reduce chemical input in conventional agricultural practices and increase nutrient uptake and stress resilience in plant species.

**Keywords:** *Zea mays* L.; environmental stresses; endophytic bacteria; plant growth promoting ability

#### **1. Introduction**

The enhancement of crop productivity is required for feeding the increasing population in developing countries and often relies on the use of chemical fertilizers. However, long-term use of these fertilizers was shown to decrease bacterial diversity in soil [1,2] and can also have harmful effects on the environment, such as leaching of phosphorus and nitrogen into groundwater, and increasing soil and groundwater pollution [3]. One way to increase the sustainability of agricultural practices is the use of efficient, nutrient mobilizing microorganisms to reduce the need and dependency on chemical fertilizers [4,5]. Plant growth promoting bacteria (PGPB) that form symbiotic interactions with their host plants are crucial to improve plant productivity and health under various environmental conditions [4,6–8]. Bacterial endophytes colonize plant tissues without any apparent pathogenic symptoms and establish beneficial associations with their plant host through phytohormone synthesis, enzyme production, and nutrient mobilization and translocation, such as phosphate (PO4 −3) solubilization, nitrogen fixation, and ammonia (NH3) production [9–11]. Moreover, many endophytes display various applications such as antimicrobial mechanisms, which reduce crop losses caused by pathogens [12–16], and its metabolites integrated into different biotechnological applications [17–20].

The Sinai Peninsula is located in the Sahara-Arabian deserts and represents approximately 6% of the total land area of Egypt. The semi-arid to arid climate and winter precipitations are the main characteristics of the Sinai Peninsula desert. Plants growing in desert conditions were found to harbor a microbiome that increased the biomass during drought stress periods [21]. Medicinal plants from desert farming in Sekem (Egypt) were shown that their roots are strongly associated with bacteria [22–24]. Although the Sinai desert has diverse medicinal plants, very few studies have focused on the associated bacterial endophytes and their PGP activities. Hanna et al. [25] collect 43 different plant species from the North Sinai desert, and reported that *Fagonia mollis* was the highest plant species harboring culturable bacteria. Among these bacteria, *Gluconacetobacter diazotrophicus* was the lowest endophytic species exhibit N2-fixing activity. In the same regards, 132 endophytic strains were isolated from 18 Egyptian medicinal plants, including nine fungal strains isolated from *Achillea fragrantissima* and exhibiting inhibitory activities against different pathogenic bacteria and yeasts [26]. Application of the bacterial endophytes (*Bacillus thuringiensis*) led to improved plant growth and increased relative water content, chlorophyll content, chlorophyll fluorescence parameter (Fv/Fm ratio), and fruit yield of sweet pepper plants [27].

Maize has become a staple food in many parts of the world, with the total production of maize surpassing that of wheat or rice. Maize crop has several uses, such as food stuff for human or as animal feed because of its high nutritional value. Maize has also been used for corn ethanol and other maize products, such as fructose, corn starch, corn oil, and corn syrup [28]. The Arab Republic of Egypt is the largest country consuming maize at the level of the African continent. However, the production of the Arab Republic of Egypt reached to about 1% of the total global production during (2005–2013), while Egypt represents the third place at the level of the African continent [29]. Therefore, the current study aimed to improve the performance maize growth under the optimal conditions and at normal habitat.

*Fagonia mollis* and *Achillea fragrantissima* are medicinal plants frequently found in the Sinai Peninsula, and their bacterial endophytes could partially be responsible for the production of various bioactive compounds [30], and the ability of these plants to withstand the harsh, drought condition of the Sinai Peninsula. Therefore, this study focused on the isolation and characterization of putative bacterial endophytes from *F. mollis* and *A. fragrantissima*, which are native inhabitants of the arid and extremely harsh conditions of the Sinai desert. Plant growth promoting (PGP) properties of the bacterial endophytes involving extracellular enzymes (amylase, cellulase, protease, pectinase, and xylanase) production, antimicrobial activity against selected pathogenic bacteria and fungi, indole-3-acetic acid (IAA) and NH3 production, and P-solubilization ability were evaluated. In addition, their effect on maize growth, plant biomass production, and nutrients content in plant shoots were also investigated in order to evaluate their potentials as bioinoculants for sustainable agriculture practices.

#### **2. Materials and Methods**
