The Improvement of Morphological, Biochemical and Antioxidant Characterizes in Mexican lime Seedlings, Inoculated by Fungal Endophyte Extracted from Green Seaweed

Introduction  Fungal 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 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 that limiting continuity of citrus production. Lack of suitable soil, is one of 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). Here, we have evaluated the potential of inoculating Mexican lime seedlings with seaweeds fungi endophyte, Aspergillus niger, to improve morphological, biochemical, antioxidant and photosynthesis pigments characterizes. 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. Fungi 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 for chemical studies. The purpose of this study was the isolation of associated fungi with seaweed species in Persian Gulf to investigate morphological and molecular characterization by using PCR amplifications ITS1-5.8S-ITS4 regions and primitive assessment of their potential as bio-fertilizer. Materials and Methods  The main aim of this study was investigation the role of endophytic fungi (Aspergillus niger), in improving the growth of Mexican lime seedlings. Cladophoropsis membranacea, green seaweed, was collected from coastal region of Bushehr province. Fungal endophytes were isolated and identified based on morphological and molecular methods. Molecular characteristic was investigated using PCR amplification of ITS1-5.8S-ITS4 regions. 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, Hormozan University. Isolated fungi by MT420720 accession number was used as bio-fertilizing agents in eight months old Mexican lime seedlings. The suspension was adjusted to a concentration of 1×106 cell per ml. For better contact of seedlings with fungi, inoculation was performed three times. After three months, morphological (trunk diameter, stem length, root length and width, leaf and branch number, leaf, stem and root dry and fresh weight), biochemical (Protein, MDA and SPAD), antioxidant (CAT, POD, SOD, APX and Gr enzymes activity) and photosynthesis pigments (Chlorophyll a, Chlorophyll b, Total Chlorophyll and Carotenoids) characterizes in treated Mexican lime seedlings were analyzed. The experiment was arranged in randomized complete design with three replications. 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 genera of Aspergillus was the most frequent isolates of the isolated fungi. The results show that most traits were significant compared with control. For example, leaf number (144.42%), root fresh weight (144.13%), stem fresh weight (94.85%) and root width (105.55%) were significantly higher compared with control (P>0.001). Fungal inoculation can significantly improve the photosynthesis pigments such as chlorophyll a (10.98%) and carotenoids (40.62%) (P>0.001) compared with control. In antioxidant capacity of seedling, CAT, POD, SOD, Gr and APX enzymes were analyzed. Fungal inoculation can increase the enzymes activity. For biochemical traits, fungal inoculation can significantly increase SPAD number and decrease MDA in inoculated seedlings compare with control (P>0.001).  Conclusion  The results showed that the use of entophytic fungi increased the growth of Mexican lime seedlings. Thereby it can be used as an effective tool for growing salinity-sensitive plants such as Mexican lime in saline conditions.