Effect of treatments (Laser or/ and Rhizobium) on morphological clover plants in irrigation with fresh water and ground water
Effect of treatments on plants hight:
For using fresh water, the results were indicated that plant heights of clover plants were increased of (29.5, 31.3, 32, 33 and 33.5 cm), (33.5, 35.3, 36.7, 38.8 and 41 cm), (45, 50, 51.5, 52.5, and 55.5 cm) , and (55, 55.5, 56, 58.8 and 60 cm) for (N2, Rhizobia, N2+Laser, N2+Rhizobia, and Rhizobia+Laser) for first, second, third and fourth cutting of clover plants, respectively (Fig. 3).
For using ground water, the results were indicated that plant heights of clover plants were increased of (21.5, 24.5, 27.7, 28.8 and 31 cm), (32, 32.3, 33.7, 36.3 and 38.4 cm), (41, 44, 48, 50 and 51.5 cm) , and (51.5, 52.5, 54, 55, and 56 cm) for (N2, Rhizobia, N2+Laser, N2+Rhizobia, and Rhizobia+Laser) for first, second, third and fourth cutting of clover plants, respectively (Fig. 4).
Inoculation with diazotrophic bacteria like Rhizobium enhanced the plant growth as a result of their ability to fix atmospheric nitrogen (Yadav and Verma, 2014). Basu et al. (2011) reported that Rhizobium inoculation was more effective for increasing the growth traits, yield and oil content of Arachis hypogaea.
Deshwal et al. (2010) concluded that the use of rhizobia inoculant enhanced plant growth in Mucuna plant.Not only Growth parameters were significantly influenced by biological and chemical fertilization but also it depended on growth stages.
Therfore, the most important finding is that Rhizobium inoculation improve growth parameters compared to nitrogen fertilization only at Advanced growth stage for “plant hight”; “Plant leaf area” and “Shoot dry weight”).
Uninoculated plants were not able to form efficient nodules as those that had been inoculated at all plant stages. Nitrogen fertilization has no significant effect on nodulation (number and weight) at different growth stages. Conversely, Rhizobium inoculated plants showed a significant high mean number and weight of nodules per plant. Rajendran et al., (2008)
The reduction in plumule length as a result of drought stress is due to plant breathing disorders in initial germination and photosynthesis in bifoliate stages. These results are in accordance with the findings of Hamidi [2000]. Data indicated that the ratio of radicle to plumule length was increased by increasing drought stress. The ratio of radicle length to plumule length is a flexibility trait which it was increased as the result of drought stress [Shalhevet, J., 1993].
German et al., (2000) observed that associative nitrogen fixing bacteria and phosphate solubilizing bacteria play a positive role in promoting the increase of root length, plant size and root surface area, as well as the assimilation of water and nutrient, and help to strengthen the respiration in root .
According to Chen et al. (2005) for example, He-Ne laser pretreatment can improve the inner energy of seeds, lead to an enhancement of cotyledon enzymes and speed up the metabolism of the cell, significantly increased the cycles of cell division (mitosis) which results in an increase in the length of the plant organs during the early growth.
Effect of treatments on Shoot numbers of clover plants:
For using fresh water, the results were indicated that the shoot numbers of clover plants were increased of (7, 8, 9, 10 and 12), (10, 11, 12, 13 and 14), (14, 15, 16, 17 and 18) , and (18, 19, 20, 21 and 22) for (N2, Rhizobia, N2+Laser, N2+Rhizobia, and Rhizobia+Laser) for first, second, third and fourth cutting of clover plants, respectively (Fig. 5).
For using ground water, the results were indicated that the shoot numbers of clover plants were increased of (5, 6, 7, 8 and 9), (7, 8, 9, 10 and 12), (12, 13, 14, 15 and 16), and (16, 17, 18, 19 and 20) for (N2, Rhizobia, N2+Laser, N2+Rhizobia, and Rhizobia+Laser) for first, second, third and fourth cutting of clover plants, respectively (Fig. 6).
At each time point measured after each cutting clover plants, all parameters of SNF efficiency, including the number of shoots, the plant heights, the number of nodules, nitrogenase activity, and plant yields, were consistently highest for laser and the _Rhiz-positive control group and lowest for laser or and the _Rhiz uninoculated negative control group. The above parameters were measured at 6 weeks after cutting of clover plants. Results are mean values for four replicate populations of water used for irrigation of crops at fresh water and groundwater.
In the present study, we have noted the significant improvement in both amylases and protease activity in shoot and root of laser irradiated seedlings. The a-amylase activity was increased over control in both shoot and root and maximum activity was observed at 25 J cm)2. The b-amylase activity also shows similar trends with a-amylase activity and the maximum activity was noted at 20 J cm)2 in shoots and roots. The protease activity also showed increasing trends over control. Chen et al. (2005b) have demonstrated increase in amylases, transaminase and proteinases from the cotyledon of Isatis indigotica treated with He–Ne laser and microwave irradiation
Examining the effect of seed irradiation time on the elongation growth of triticale seedlings (expressed in cm units and as % of control by using the IP index) revealed that, the longest seedlings were found among grains irradiated for 3 h In these cases, the He-Ne laser had a positive effect on the number of shoots, elongation growth, and dry mass [Govil et al., 2006].
In general, the red light of the laser stimulated the morphology and physiology of seedlings and plants, although, for some features, long exposure to red light caused a slight reduction effect. The positive effect of laser radiation on plant growth and development can be caused by the ‘excitement’ of the bio-energetic structure by the formation of cells with excess energy and an increase in bio-energy levels in organisms [Vasilevski, 2003].
The growth characters, viz., length, fresh and dry weight of shoots and roots, number of leaves and roots were higher at lower doses of laser irradiation was found to be decreased at higher doses. Similarly, the dry weight of roots, number of leaves and roots were also significantly higher in lower doses of laser radiation compared with un-irradiated control. Chen et al. (2005b).
Effect of treatments on nodules number conformation of clover plants:
For using fresh water, the results were indicated that the nodules number conformation of clover root plants were increased of (50, 55, 65, 72 and 75) , (60, 67, 72, 74 and 78) , (65, 73, 80, 91 and 98) , and (91, 99, 115, 121 and 131) for ( N2, Rhizobia, N2+Laser, N2+Rhizobia, and Rhizobia+Laser) for first, second, third and fourth cutting of clover plants, respectively (Fig. 7).
For using ground water, the results were indicated that the nodules number conformation of clover root plants were increased of (30, 40, 45, 55 and 65) , (48, 55, 60, 65 and 78) , (56, 67, 73, 84 and 90) , and (86, 90, 94, 98 and 105) for ( N2, Rhizobia, N2+Laser, N2+Rhizobia, and Rhizobia+Laser) for first, second, third and fourth cutting of clover plants, respectively (Fig. 8).
Furthermore, both rhizobia and alfalfa nodules are most abundant in the 30- to 60-cm soil layer, possibly because nodules in this region are the most actively fixing.
In the present study, plant response to the different inoculant formulations differed among treated seeds by rhizobiz or and laser treatments and untreated seeds.
Pii et al. (2007) reported that Rhizobium inoculation increased nodule number on Medicagospp. Yadav and Verma (2014) stated that the dual combinations of Rhizobiumleguminosarum either with Pseudomonasaeruginosa, Bacillus megaterium and Azobacterchroococcum, significantly enthused nodules number and dry weight of nodules. Granular inoculants placed below the seed encourage nodulation on lateral roots in both pulse and forage legume crops rather than the clusters of nodules in the crown of the plant that is often observed with seed applied inoculants (Kyei-Boahen et al. 2002).
In fact, the non-treated plants exhibited fewer nodules numbers throughout the course of experiments, the same results obtained from the un-inoculated _Rhiz-negative control plants. These data indicate that treatment with rhizobia and laser is sufficient to significantly nodule formation. Because nodule formation is necessary for symbiosis and the site of SNF.
It was noticed in Fig. , the nodules number conformation of clover root plants were increased by using fresh water comparing in ground water in same treatments. Also, nodules numbers were gradually increased after first, second, third and fourth cutting of clover plants. There is a significant difference was shown between the un-inoculated plants (nitrogen fertilizer) and the Rhizobium or and Laser of inoculated plants concerning the nodule number per plant at all plant cutting stages (Four cutting harvesting). (Shirin et al., 2017).
In fact, the greatest usually apprehensive modes of action of PGPR were phytohormone production, which improves plant development and growth (Vacheron et al., 2013). Possible coaction of Rhizobium bacteria inoculum and PGPR hosted in Goubellat soil could increase nodule number and nodule dry weight. Selecting efficient Rhizobium strains under greenhouse and field conditions for lentil, pigeon pea, clover and Chickpea increase nodule dry weight and shoot dry weight (Icgen et al., 2002).
Uninoculated plants were not able to form efficient nodules as those that had been inoculated at all plant stages. Nitrogen fertilization has no significant effect on nodulation (number and weight) at different growth stages. Conversely, Rhizobium inoculated plants showed a high mean nodule number per plant and a significant high mean nodule weight per plant. (Rajendran et al., 2008).
Effect of treatments on shoots and roots dry weight per twenty plants:
Effect of treatments on shoots dry weight per twenty plants:
For using fresh water, the results were indicated that the shoot dry weight (SDW) of clover plants were increased of (9.92, 10.40, 11.00, 12.19 and 14.92 g) , (9.99, 12.23, 14.60, 15.55 and 16.88 g) , (11.23, 13.55, 15.65, 17.89 and 19.34 g) , and (13.55, 14.61, 16.89, 18.99 and 22.33 g) for ( N2, Rhizobia, N2+Laser, N2+Rhizobia, and Rhizobia+Laser) for first, second, third and fourth cutting of clover plants, respectively. (Fig. 9). While, the results were indicated that the shoot dry weight (SDW)of clover plants were increased of (9.22, 9.56, 10.63, 11.23 and 12.54 g) , (9.56, 9.89, 11.59, 13.56 and 14.25 g) , (10.11, 10.55, 11.85, 13.99 and 15.44 g) , and (12.67, 13.55, 15.34, 17.35 and 19.25 g) for ( N2, Rhizobia, N2+Laser, N2+Rhizobia, and Rhizobia+Laser) for first, second, third and fourth cutting of clover plants, respectively, for shoot dry weight (SDW) at irrigation by ground water. (Fig. 10).
Alfalfa seedlings were found obviously promoted after being inoculated with LW135 or RSN219. Shoot height, root length, root volume, individual number of leaves per plant, biomass and P uptake percentage of the two varieties were found remarkably increased than control group (CK1). This shows that after the inoculation of phosphate solubilizing Rhizobium and Klebsiella bacteria the productivity and growth of alfalfa were efficiently promoted in soil that is deficient in nitrogen and available phosphorus. (Shirin et al., 2017).
As plant height and shoots number, shoot dry weight (SDW) for Rhizobium or and Laser inoculated plants was significantly higher than the uninoculated check (nitrogen fertilized and or laser). Shoot dry mass, and N uptake in above-ground tissue were measured for first, second, third, and fourth cutting at the irrigation of both fresh water and groundwater during season cut. Shoot dry mass production was very low in untreated seeds compared to treated seeds by rhizobia and laser.
In the pot experiment, Rhizobium inoculation and all nitrogen were increased the height of plant. None of the treatments had any effect on the number of leaves/plant. Also, all nitrogen were increased shoot fresh weight with or without Rhizobium. Rhizobium significantly increased the fresh and dry weight of shoot and root. Rhizobium inoculation also increased the number of nodules per plant. Nitrogen, with or without Rhizobium, significantly increased the number of nodules per plant up to 80 kg N/ha.[ Elnesairy et al., 2005]
The presence of a few weak nodules on the roots of uninoculated alfalfa plants indicates the presence of indigenous Rhizobium meliloti strains in Shambat soil, probably due to earlier inoculants applied in this area. The effect of Rhizobium in improving plant height, shoot fresh and dry weight, root fresh and dry weight, number of nodules per plant and plant density confirms earlier work such as Abuswar and Mohamed (1997).
Among the growth characteristics, length, fresh weight of shoot, dry weight of root, number of leaves and roots were higher at 25 J cm)2 whereas the fresh weight of root and dry weight of shoot was higher at 20 J cm)2 laser irradiation. The laser irradiation induced significant improvement in seed germination and subsequent growth of seedlings Chen et al. (2005b).
Effect of treatments on roots dry weight per twenty plants:
For using fresh water, the results were indicated that the root dry weight (RDW) of clover plants were increased of (0.23, 0.24, 0.26, 0.27 and 0.37 g) , (0.36, 0.39, 0.40, 0.52 and 0.66 g) , (0.48, 0.69, 0.76, 0.87 and 1.00 g) , and (0.77, 0.96, 1.11, 1.26 and 1.45 g) for ( N2, Rhizobia, N2+Laser, N2+Rhizobia, and Rhizobia+Laser) for first, second, third and fourth cutting of clover plants, respectively. (Fig. 11). While, there were increased of of (0.16, 0.20, 0.22, 0.23 and 0.31 g) , (0.31, 0.33, 0.36, 0.41 and 0.51 g) , (0.39, 0.41, 0.65, 0.81 and 0.96 g) , and (0.59, 0.67, 0.79, 0.99 and 1.23 g) , respectively for root dry weight (RDW) at irrigation by ground water. (Fig. 12).
Positive correlation have been found between root biomass and dry mass yield in alfalfa (Hakl et al., 2008). According to Kusvuran et al. (2014) the amount of nitrogen accumulated in alfalfa roots were observed to be much higher than that for other legumes.
Carbon and nitrogen content are good indicators for soil quality. Plants can translocate 20-50% of total fixed carbon to their roots (Kuzyakov and Domanski, 2000). With the maximizing formation of root biomass C inputs into the soil increased. Root biomass contains approximately 40% С and about 18% of the root С for the year could be converted into humus (Kwabiah et al., 2005).
Laser irradiation and Rhizobium inoculation of seeds significantly increased plant height compared to the uninoculated plants (Nitrogen fertilization and or Laser). Plant height was significantly enhanced under Rhizobium inoculation in comparison with nitrogen fertilization.
Vessey and Heisinger, (2001) also found that the accumulation of dry matter in pea was increased after the inoculation with a phosphate solubilizing fungus Penicillium bilaii. Rice and Lupwayi (2000) found that the cooperation of phosphate solubilizing bacteria and Rhizobium was good to increase the yield of crops. Combined inoculations with N2-fixing and P-solubilizing bacteria were more effective than single inoculation (Erdal et al., 2008).
Long irradiation times and high wavelength can cause considerable damage to the seed structures. The physical phenomenon can be observed in the case of seeds when they first absorb the radiation energy and then transform it into chemical energy and use it for subsequent growth [Abdullateef and Osman, 2011].
In the recent times, pre-sowing stimulation of seeds with laser light has become a popular method of improving the sowing material. This method is especially popular in organic farming, as it is safe for the natural environment (Hernandez et al. 2010).
Several studies performed on various crop plants showed that the effects of laser stimulation can depend on many factors: the wavelength, dose, exposure time, the species and cultivar of plant (Ry- biński 2001).
Govil et al. (2006) reported the maximum increase in shoot and root length, dry weight of the seedlings from laser irradiated (30 min) green gram seeds., and enhanced emergence, early flowering and maturity, plant height due to the presowing He–Ne laser treatment than the control plants of faba bean.