Isolation and Identification of taxonomically diverse bacterial endophytes from citrus in Punjab Pakistan

Citrus is an economically important fruit crop grown in all provinces of Pakistan, while Punjab accounting for 95 percent of total production due to its favorable climate for citrus production. Commercially grown varieties in Pakistan include sweet oranges, grapefruits, Mandarine, Lime, and lemon. The goal of this research was to see how diverse the cultivable bacterial populations are found in citrus cultivars. Out of 90 isolated cultures, 37 endophytic bacterial species and 15 different genera of bacteria were characterized based on morphological, biochemical, and molecular methods from citrus leaves. All the isolated bacteria were subjected to PCR amplification through 16S rDNA followed by sequencing. RDP base classification revealed that class Bacilli has the largest percentage of isolates, whereas class Alpha, Beta, and Gamma Proteobacteria have the lowest percentage among all genotypes used. According to the findings, the phylum Firmicutes contains a common genus (Brevibacterium, 1%; Enterococcus, 6%; Staphylococcus, 7% and Bacillus, 60%). Alpha (Rhizobium) beta (Burkholderia cepacia; Comamonas terigena) gamma Proteobacteria (Enterobacter hermachei (1%), Klebsiella pneumoniae (1%), Proteus mirabilis (8%), Pseudomonas aeruginosa (5%), Psychrobacter pulmonis and Yersinia molalretti (1%) respectively. These results revealed that cultivars of the plants might contribute to the structure and endophytic bacterial communities associated with citrus. Endophytes extracted from leaf samples of different citrus cultivars in Pakistan are reported for the first time. The idea of employing endophytes bacteria to produce enzymes stimulate plant growth, and its purpose as a biological control agent will be investigated in the future.


Introduction
Citrus has traditionally been recognized as one of the most popular fruit, as it is enriched 46 with minerals, vitamins A, B, and C, ascorbic acid, and also possesses strong antioxidant 47 potential [1,2]. Pakistan is the world's number one producer of Kinnow mandarin [3]. A 48 widespread kind of microorganisms exists in the phyllosphere (Surface of the plant). 49 Bacteria are the most common microbes found in the phyllosphere. Endophytes (bacteria 50 that live inside plant tissues) and epiphytes (bacteria that live on the surface of plants) are 51 both examples of phyllosphere bacteria [4,5]. The research on plant-based endophytes is 52 critical for understanding the diverse bacterial interactions that occur in certain 53 environments, which help to improve their biotechnological applications [6,7]. The 54 endophytes that reside within the surface of the leaf form complex population dynamics 55 that are important for agriculture and the environment. Plant health can be improved or 56 arrested by the bacterial diversity associated with the leaf, which can also drive the 57 colonization and infection of tissues by phytopathogens [8,9]. 58 Microorganisms that live in a mutualistic connection with a plant are known as 59 endophytes [10,11]. Plant processes such as growth promotion, nutrient uptake, abiotic 60 stress tolerance, and pathogen infection inhibition are thought to be supported by them 61 [6]. Bacterial endophytes (BE) are found in different parts of plants such as roots, stems, 62 and leaves [12,13]. The population density of BE is affected by multiple factors, 63 including the plant's developmental phase [7,14], cultivar (genotype) [14,15], the 64 portion under examination [16], comprising the type of plant being studied, and the interaction between microbes and ecological factors [17]. The population density of BE 66 could be 10 2 to 10 9 people per square kilometer [18]. Commonly, BE present in lower 67 numbers as compared to the rhizospheric bacteria [19]. They are not limited to a single 68 species in a single plant host, but they could be observed in multiple genera and species. 69 Endophytic bacteria have been extracted from different segments of M. micrantha, 70 including the roots, lamina, and petiole [20]. In previous studies, endophytes have been 71 isolated from cottonwood (Populus deltoids) [21], grapevine (Viti vinifera) [22], poplar 72 (Populus alba) [23], sweet potato (Ipomoea batatas) [24], potato [25,26], soybean 73 (Glycine max) [27] and tomato (Solanum lycopersicum) [28]. 74 Pakistan has an agriculturally dominated economy with large areas of fertile land and a 75 diversified geographical region and climate. There has been limited research on microbial 76 biodiversity, and no such type of research on the diversity of BE from Citrus in Pakistan 77 has ever been reported. It is so believed that bacterial species found in Pakistan carrying a 78 wide variety of endophytic bacteria will be investigated to find the real picture. This 79 study will provide information regarding the different strains of bacteria that are residing 80 in citrus leaves and still need to be identified from different geographical locations of the 81 country. Database and attained their Accession numbers (Table 3).   (Table 1 and 2).

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Note: Pos. and Neg. Abbreviations represent the positive and Negative respectively.

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To get a representative sample of the cultivable endophytes living inside leaf tissues The results showed that the majority of genera belonged to the phylum Firmicutes made 159 up of genus (Bacillus accounts for 60%, Enterococcus for 6%, Staphylococcus for 7%, 160 and Brevibacterium for 1%). Additionally, during this study, the phylum Proteobacteria and Accession no., are presented in the table below (Table 3).   (Table 4).

Molecular characterization of bacterial endophytes
The search for novel bacterial endophytes may contribute to the discovery of novel processes that boost plant growth and disclose interesting interactions between plants and bacterial endophytes. Though the use of 16S rDNA sequencing to identify bacterial diversity has resulted in the recognition and characterization of many previously unknown bacteria, determining pathogenic microbes and correlating them to disease still requires bacterial cultures.
[49] Conducted similar studies on the 16S rRNA from the soya bean. However, in beans, 16S rRNA sequencing was performed, and a taxonomic study was applied to determine the evolutionary pattern between bacterial strains [50].

Signature Sequences
Signature patterns of all isolated genera were identified as signature patterns are critical