Saffron is an aromatic plant belonging to the Iridaceae family and is produced from the dried stigmas of Crocus sativus [1]. Crocus sativus is a triploid herbaceous geophyte propagated using alternative bulbs, which are grown in environments with very different soil characteristics that are used worldwide as a natural spice and color, due to its particular red stigma [2, 3].
The genus Crocus (family: Iridaceae) consists of about 80 different species, which are cultivated worldwide for medicinal purposes. C.sativus is produced worldwide, mainly in Spain, India, Turkey, Greece, Austria, Belgium, France, Germany, the Netherlands, Italy, Japan, Norway, Russia, Switzerland, Turkey, China, and Iran [4].
The organoleptic properties of this spice are attributed to the relative percentage of its secondary metabolites, such as crocin, picrocrocin, and safranal, which provide color, taste, and aroma, respectively. The concentrations of these compounds are combined to determine the quality of Crocus sativus spices, as defined by the International Organization for Standardization[5] .
Since Crocus sativus requires a lot of effort to harvest the flower daily and separate the stigma, it has a low yield, and increasing its quality can be very profitable. In this regard, adopting sustainable culture techniques such as biological materials may further help increase the yield of spices and the accumulation of active elements [6].
Microorganisms living in almost every conceivable environment in the biosphere play unique and integral roles in the ecosystem. The microbiome is the sum of the microbes associated with different plants, often referred to as the plant's second genome. Some microbes, such as plant growth-promoting microbes (PGPM), have positive interactions, while microbial pathogens interact negatively with plants. Likewise, microorganisms in the soil are one of the main factors affecting the production and quality of Crocus sativus.
A study aimed at manipulating the plant microbiome to suppress pathogen populations in wheat showed that strategic management has a negligible effect on the microbiome compared to geographical location. Also, the variety of bacteria in wheat root was more than in its stem and leaf, but the type of fungi in the stem is more [7].
The structure of the microbial community changes as we move from the rhizosphere to the endosphere. However, there are positive and negative effects in all areas. There is generally a broad scope for the widespread use of these microbial communities to improve the environment. Recent research has focused on the benefits of soil organisms for crops, significantly increasing plant nutrient uptake. The soil is not only the site of the plant life cycle but also the main reservoir of a wide range of microorganisms such as bacteria and fungi.
With a scarcity of arable land resources, plant-microbial interaction as coexistence can enable plant survival to overcome the challenges of environmental stress, including low-nutrient soils with little water. Given the interaction of plants and natural microbes to tolerate stress and plant stress, coexistence-based technologies are created for fertilizer production[8]. Bacterial microbiome manipulation also enables scientists to influence beneficial plant activities such as growth restriction, plant defense, plant growth, and plant health, and potentially reduce the use of chemical fertilizers. In this study, it has been stated that the reason why the soil microbiome should be changed for the plant is that never all of the soil microbiomes are used for the plant; as a result, a limited number is beneficial for the plant, and changes the physiology of the plant and increases the plant's resistance to pathogens and increases crop yield. Also, the content of materials used by each plant is different [9].
The 16srRNA gene is part of the ribosome that translates mRNA. This gene is found in all bacteria and primates, while other genes are not found in all living organisms. The 16srRNA gene contains highly conserved regions for general primer design in PCR. Specific primers for the 16srRNA gene are also available. This gene consists of 9 areas (V1-V9) that are currently the most widely used parts of V1-V3, V4, and V4-V5. Because research has shown that more accurate sequences can be seen from these areas. Also, using these sequences, it is possible to compare sequences and determine the taxonomy of organisms[10] .
In general, fungi are less susceptible to drought than bacteria due to their carbon sequestration. Also, gram-positive bacteria are less affected by dehydration conditions than gram-negative bacteria due to having a strong cell wall. Scientists studied microbes using the analysis of phospholipid fatty acids (PLFA). The results showed that with decreasing rainfall, the microbial community decreases, and this decrease is more severe in bacteria than in fungi. Bacterial size is also directly related to soil nitrogen [11]. In a study aimed at identifying the fungal endophytic population associated with Crocus sativus and identifying the microbial formula useful for sustainable culture, after sampling, fungal endophytes were extracted from Crocus sativus bulbs and then propagated on a culture medium. In the next step, its DNA was extracted, and it is ribosomal ITS gene was sequenced. Finally, MEGA6 software was used for phylogenetic identification. The results showed that P. mustea and C. maloru had the highest content on the Crocus sativus bulb [12] .
Soil sampling is generally done mainly from the rhizosphere; because scientists have stated in their research that the rate of bacterial diversity in the rhizosphere is 40 times that of non-rhizosphere soil in the topsoil [13]. Also, in a study examining the relationship between bacteria and plants (rhizosphere) in the Mediterranean region in occupied Palestine, soil samples of native plants (Rhamnus puncata, Calicotome villosa, Sarcopoterium spinosum, Quercus ithaburensis) were harvested in the shade and sunlight. The DNA of their bacteria was then extracted and sequenced using the Miseq method. Finally, the relative abundance of bacteria and their types were investigated. The results showed that organic matter and soil moisture play a vital role in the structure of the bacterial community. PerMANOVA software also showed that the bacterial community causes significant differences between plants in different regions [14].
In another study, the Miseq method was used to identify beneficial microorganisms in Crocus sativus. In this method, the samples were sequenced and analyzed by bioinformatics software. In this study, it was found that Nectriaceae, Penicillium Aspergillus, and Saccharomycetales can have a positive effect on Crocus sativus bulb germination [15] .
Considering the economic importance of Crocus sativus in Iran and other parts of the world, increasing its quality compared to the current product is of great importance. Since Ghayen Crocus sativus has a higher quality than other cities in Iran [16] and in Ghayen city, the quality of the Shahik region is higher than other regions [17]. However, all climatic conditions, as well as the soil texture of the region, are the same in the Crocus sativus farms of the Ghayen region [18] .
The western heights of Ghayenat city (33° 43′ 48″ N, 59° 10′ 48″ E) are in the northwest and southeast directions. The most important mountain in this range is Siahkooh. The most important feature of these heights is the creation of cold and humid air in this city, which provides the conditions for Crocus sativus cultivation and differentiates the quality of Crocus sativus in the Ghayen region from other regions.
The plains located in the northern part of Ghayen city (Shahik region) (Fig. 1) are very rich in agriculture and Crocus sativus cultivation. But other Ghayen plains do not have this feature, and this is while all climatic conditions, as well as the type of soil texture in the region, are the same [18].
Even though all climatic conditions are the same, the quality of Crocus sativus in the Shahik region is different from other regions. It has better quality indices (picrocrocin (taste), safranal (aroma), and crocin (color)). This study aimed to investigate and compare the soil microbiome of Shahik Crocus sativus root with other regions and identify the microbiome that affects the quality of Crocus sativus. In this study, considering all the conditions for Crocus sativus cultivation are the same, considering the possibility of microbiome differences in different regions and its effect on Crocus sativus quality, we examined the Crocus sativus microbiome in these regions.