Symbiosis of Algal Endophytes Aspergillus niger and Bacillus aquimaris OD14 with Mexican Lime Seedlings under Salinity Stress Condition

Introduction Endophytes have symbiosis life within the plant tissues without causing any obvious negative effects. Seaweeds are one of the large and diverse groups of marine plants that play an essential role in marine and oceans ecosystems. Seaweeds show rich diversity of associated microorganisms in compare with the other multicellular organisms. Citrus species, are amongst the most important evergreen fruit trees, cultivated in many countries worldwide. There are several obstacles for citrus production in southern of Iran, limiting continuity of citrus production. Lack of suitable soil, salinity and drought stresses are the main challenges threatening citrus industry in southern of Iran. Similar to other citrus species, the production of Mexican lime is threatened by certain biological stresses (such as pests, plant diseases and weeds) and non-biological stresses (such as salinity, drought, floods, cold and heat stress). Endophytes are advantageous group of microorganisms that protect plants from biotic and abiotic stresses. One of the alternative ways to restore normal plant growth may be to use plant growth to stimulate endophytes. Endophytes can play an important role in plant growth. Endophytes from marine environment are gaining special interest because of their existence in the harsh conditions of marines and ocean ecosystem such as temperature, light availability, high salinity and osmotic stress. Endophytes have already been isolated from various marine habitat, including marine plants, marine invertebrates and vertebrates. Among these organisms, seaweeds are one of the most prevalent sources of marine-derived fungi and bacteria for chemical studies. The purpose of this study was the isolation of associated fungi and bacteria endophytes with seaweed species in Persian Gulf to investigate morphological and molecular characterization by using PCR amplifications ITS1-5.8S-ITS4 regions and 16s rRNA gene respectively. Here, we have evaluated the potential of inoculating Mexican lime seedlings with seaweeds fungi and bacteria endophyte combination, (Aspergillus niger+ Bacillus aquimaris OD14), to improve morphological, biochemical, antioxidant and photosynthesis pigments characterizes of Mexican lime in salinity condition.   Materials and Methods The main aim of this study was to investigate the role of endophytic fungi (Aspergillus niger) and bacteria (Bacillus aquimaris OD14) in improving the growth of Mexican lime seedlings. The seaweed samples were collected from coastal regions of Bushehr province and Qeshm Island. Fungi and bacteria endophytes were isolated and identified base on morphological and molecular methods. Molecular characterization was investigated using PCR amplification of ITS1-5.8S-ITS4 regions and 16s rRNA gene respectively. Mexican lime seeds were sterilized with 0.5% sodium hypochlorite for 20 minutes and then completely distilled three times with distilled water. Seedlings pots containing autoclaved soil were placed in the greenhouse of the Faculty of Agriculture, Hormozgan University. The experiment was arranged as a factorial experiment based on randomized complete randomize design with three replications. Isolated fungi and bacteria by MT420720 and MT278260 accession numbers were used in eight months old Mexican lime seedlings. The suspension was adjusted to a concentration of 1×106 cell per ml for fungi and 1×108 cell per ml for bacteria inoculums. For better contact of seedlings with endophytes, inoculation was performed three times. After three months, salinity stress was applied. morphological (Leave, Stem and Root dry and fresh weight), biochemical (Protein, MDA and soluble sugars), antioxidant capacity (CAT, POD, SOD, APX and Gr activity) and photosynthesis pigments (Chlorophyll a, Chlorophyll b, Total Chlorophyll and Carotenoids) characteristics in treated Mexican lime seedlings and control were analyzed. Analysis of variance of traits was performed using SAS software version 9.4 and the means were compared using LSD method with a probability level of P≤0.05.   Results and Discussion The results show that most characterizes were significant compare with control. For example, in 6000 µs/cm water salinity, leave fresh weight (203.49%), root fresh weight (347.41%), stem fresh weight (206.81%) and root dry weight (421.95%) were significantly higher compared with control (P>0.001). Endophytes inoculation can significantly improve photosynthesis pigments such as chlorophyll a (65.21%), chlorophyll b (11.9%), total chlorophyll (28.39%) and carotenoids (59.09%) (P>0.001) compare with control. In antioxidant capacity of seedling, CAT, POD, SOD, Gr and APX were analyzed, Endophytes can increase enzymes activity. For biochemical characterizes, in 6000 µs/cm water salinity, endophytes can significantly increase soluble sugars (17.85%) and decrease MDA (35.18%) in inoculated seedlings compare with control (P>0.001).    Conclusion The results showed that the use of endophytic fungi and bacteria can increase the growth of Mexican lime seedlings under salinity stress. Thereby it can be used as an effective tool for growing salinity-sensitive plants in saline conditions.