Detection of Family Specialty for Microbacterium sp. AJ-Z Isolated from Fenugreek ( Trigonella foenum-graecum ) Root Nodules

Microbacterium sp. AJ-Z was isolated from fenugreek ( Trigonella foenum-graecum ) root nodules on Yeast Extract Mannitol (YEM) medium. Colonies of the bacteria isolates grown were rod shape, whitish, translucent, sticky nature. The results showed responded of the fenugreek seedlings grown on Nitrogen free medium (NF) to inoculated with Microbacterium sp. AJ-Z isolate at 30 min. and successful to form nodules its roots in the rate of 70% after 7 days with rate number of nodes/seedling was 2.9 superiority on the other which grown on Murashige and Skoog (MS) medium and 0.7% agar. Nodules produced have a spherical shape, pinkish and indeterminate in growth and after a few days, its shape becomes elongated.


INTRODUCTION
The fenugreek (Trigonella foenum-graecum L.) belongs to Leguminoceae family (Seasotiya et al., 2014), cultivated in the world for leafy vegetables, condiments and medical (Paul and Pal, 2014). Dry fenugreek seeds have been used in health benefits, such as diuretics, antibacterial, anti-oxidant, anti-tumor and diabetes (Moradi-kor et al., 2013). The plants of Leguminoceae have important role in increase the fertility of the soil and nitrogen fixation through its symbiosis relationship with rhizobium (Pervin et al., 2017). The fixation of N 2 process by Rhizobium achieved in the root nodules by Nitrogenase that reduce atmospheric nitrogen N2 to ammonia NH 3 (Singh et al., 2008). The simultaneous infection with rhizobia and rhizosphere bacteria increase nodulation and growth in an extensive variety of legumes (Singh et al., 2013).Use of bacterial inoculums leads to increase the number of root nodules that rise the capability of nitrogen fixation (Bader et al., 2015). The inoculation of Rhizobium increasing highest benefit of the number of leaves per plant, plant dry weight and number of nodules per plant compared with the un-inoculated control (Gendy, 2013).
The genus Microbacterium belong to the phylum: Actinobacteria, the class: Actinobacteria, the order: Actinomycetales and the family: Microbacteriaceae (Hadjadj et al., 2016), which is positive for the Gram stain. Microbacterium is an endophytic bacterium that colonizes the root nodules of leguminous plants, non-symbiotic (Peng et al., 2015). Which have more than 90 species isolated from different sources such as Microbacterium phyllosphaerae were isolated from the phyllosphere of grasses (Behrendt et al., 2001). Also from different plants, such as sweet corn, Zea mays, cotton (Zakhia et al., 2006), Ornithopus compressus. andFenugreek (Qaddawi, 2021 . ( They do not symbiotically induce nodules but their coexistence for assisting nodulation ability has been proven by many studies (Martínez-Hidalgo and Hirsch, 2017;Zhang et al., 2018). The aim of this study, detection the family specially of Microbacterium AJ-Z sp. On the Fenugreek (Trigonella foenum-graecum).
Present study aimed to characterization the bacteria according to the microbial test and detection of family specialty for Microbacterium AJ-Z sp. by re-inoculation with the root of same plant.

MATERIALS AND METHODS The source of Microbacterium sp. AJ-Z
The Microbacterium AJ-Z sp. provided from plant tissue culture laboratory in department of Biology/ College of Science / University of Mosul/ Iraq, which sub cultured on the solid YEM medium every 20 day (Qaddawi and Mohammed, 2021).

Preparation of Microbacterium sp. AJ-Z cell suspension
Suspension of the Microbacterium sp. AJ-Z was prepared by taking a lob full of bacteria and placed in a vial contain 20 ml of liquid YEM medium and incubated at 28 °C for 48 hours in the shaker incubator) New Brunswick Scientific Co., Inc. Edison. N.J. USA(with100 r / min. rotational speed (Godschalx et al., 2017).

Characterization of Microbacterium sp. AJ-Z
Visual characterization of colony morphology was done for isolate of Microbacterium sp. AJ-Z isolate.

Gram's staining
Microbacterium sp. AJ-Z suspension with 48 hrs. old was tested to the Gram's staining using aqueous Crystal violet solution (0.5%) and iodine solution. Later observed under research microscope (make: Olympus) at 40X.

Surface sterilization of fenugreek (Trigonella foenum-graecum) seeds
Seeds of fenugreek (Trigonella foenum-graecum L.) plant were surface sterilized by soaked in ethanol 96% for 2 min. flowed in 3% sodium hypochlorite (NaOCl) with stirred for 5 min., then finally rinsed with sterilized distilled water three times/ one minute (Al-Mahdawe, 2013). Sterilized seeds placed on the surface of solidified Murashige and Skoog medium (MS), (Murashige and Skoog,1962), Nitrogen free medium (NF), (Fahraeus, 1957) and 0.7% agar individually. Samples maintained in culture room at24∓2 °Cin the dark. After produced the complete seedlings were transported to the same condition but in 1500 lux with 16 hours light / 8 hours dark.

Detection the family specialty of the Microbacterium sp. AJ-Z isolates
Forty of fenugreek seedlings growing with 3 days on each of MS, NF and 0.7% agar medium were inoculated individually by immersion their root in flask contain Microbacterium sp. AJ-Z suspension for15, 30 and 60 min. alone, then the seedlings were dried using sterile filter papers and transferred to the surface of 20 ml of solid NF medium in a 9.0 cm plastic petri dish at a rate of 2-3 seedlings / plate. The plates were sealed with their covers and coated with parafilm and kept vertically in the growth incubator at 24 ± 2 °C under 16 hours light / 8 hours of darkness conditions and intensity of light 2000 lux, with covering the root system with black tape.

RESULTS AND DISCUSSION Cultural characters of Microbacterium sp. AJ-Z
The colonies of the isolate Microbacterium sp. AJ-Z which grown on solid YEM medium after two days of incubation showed a sticky form, indicate the production of mucous substances., also observed as whitish, colored till 3-4 days of growth and turning yellowish in color after 4 days. This result similar to (Singh et al., 2008;Tsegaye et al., 2015) Production of nodules on the fenugreek seedling roots The results indicated for ability of Microbacterium sp. AJ-Z bacteria isolate to re-infected the roots of fenugreek seedlings when treated with them and the success of the root nodule formation process, with different ratio according to the seedling sources, which growing on MS, NF, 0.7% agar and treatment times (Table 1).

Number of seedlings inoculated: 40
The period of treatment 30 min. was the most prominent for the root nodule formation, which reached to 35%, 70%, and 45% more than other treatment times. The seedlings growing on NF medium showed a superiority in their response to the inoculation process by formed nodules with 70% with 2.9 of nodules/seedling more than other types on seedlings. The hairs of their root's appearance various changes after 3 days of inoculated with bacteria Fig. (A) represented by their transformed from a straight shape to a curved shape (Fig. B). which continued to grow and form a spherical shape represented by a nodule Fig. (C). After two weeks have elongated oval shape, it developed into a complete root node Fig. (D). Root nodules formed on fenugreek seedlings was thus successful Fig. (E). The formation of root nodules of the fenugreek seedlings when treated with Microbacterium sp. AJ-Z bacteria represents the success of the symbiotic relationship between them (Meena et al., 2018). Also, it begins with the occurrence of abnormalities in the root hairs as a result of their secretion of flavonoids that stimulate the specialized genes and encoding those genes for production the nod factor and linking its chitin part in a specialized way with the fenugreek lignin, The incidence of infection and node formation (Andrews and Andrews, 2017). A number of studies indicated the inability of the genus Microbacterium to establish a symbiotic relationship with leguminous plants (Wang et al., 2006;Palaniappan et al., 2010;Dudeja et al., 2012), while it is endophytic and only has the ability to fix atmospheric nitrogen because it possesses the nif H gene ( Gtari et al., 2012;Yadav et al., 2018). Its ability to form root nodules may explain to what Zakhia (2006) mentioned that the sequences of nitrogenous bases in the nif H gene of the bacterial genera Microbacterium, Agromyces, Starkeya and Phyllobacterium isolated from the root nodules of leguminous plants are more similar to the sequences of the nif H gene of S. meliloti as it reached The percentage of congruence according to the NCBI site between the nif H gene of S. meliloti and the nif H gene of three strains of Microbacterium, namely, ORS 1417b, ORS 1418b and ORS 1472b to 94%, 93% and 96%, respectively, and the possibility of the evolution of endophytic bacteria into symbiotic bacteria by It acquires symbiosis genes within root nodules through horizontal gene transfer, which occurs naturally in the soil when appropriate conditions are provided for the conjugation process (Taghavi et al., 2005).The superiority of the growing seedlings on the NF medium in nodule formation ratios may be explained by the fact that it does not contain nitrogen sources and thus stimulates the seedlings to be infected (Saito et al., 2014).

CONCLUSIONS
The isolated bacteria from root nodules of fenugreek were sticky, whitish color and success in forming nodules on the root of fenugreek after inoculation and superiority MS medium with 30min for inoculation on the rest media which used in this study.