Strategy Role of Mycorrhiza Inoculation on Osmotic Pressure, Chemical Constituents and Growth Yield of Maize Plant Gown under Drought Stress

The present work was carried out to investigate the role of mycorrhiza inoculation at two harvesting stages (90-days and 30-days) of maize plants grown in pot experiment with different moisture content levels 100%, 70%, 50% and 20%. Drought stress tolerant in maize plant was varied in different organs of the same plants and also varied among different stage of plant development. The sensitivity of maize plants was related with reduction of root soluble sugar, shoot and root soluble protein at 30-days of plant harvesting, and soluble sugar and soluble protein in both organs of both harvesting stages. This related with reduction in OP and lowering of water uptake which induced a marked decrease in fresh and dry matter production in shoot and root of both harvesting stages. AM inoculation increase maize tolerant to drought stress presented in increasing growth parameters, chemical constituents and minerals contents compared with untreated plants. Proline content with AM inoculation was more or less unchanged in shoot of plant harvesting at 30-days and in root of plant harvesting at 90-days. However, a marked increase was induced in plant harvesting at 30-days and in shoot of plant harvesting at 90-days. Mycorrhiza inoculation induced a significant increase in OP value either compared with corresponding level or compared with control value 100% as in plant 30-days of harvesting or compared with control only as in plant harvesting after 90-days. AM infection with different moisture content levels measured by N-acetyl glucosamine content were not affected by drought stress. Results showed also that control roots contained N-acetyl glucosamine would be attributed to mycorrhiza and other fungi naturally present in soil.


Introduction
Drought is one of the most adverse abiotic stresses on plant growth and productivity, it induced morphological, physiological and biochemical effects, reduced CO 2 assimilation, leaf area, photosynthetic pigment content, stem growth, root proliferations disturbs water use efficiency. The role of chemical substances accumulation in drought plants has been researched to understand plant tolerance to water deficit [1] [2] [3] [4]. Numerous researchers have studied plant growth promoting fungi (PGPF) attributes of rhizospheric fungi [5] [6]. Among the PGPF, species of Phoma, Penicillium, Aspergillus, Fusarium, Trichoderma, and arbuscular mycorrhizal fungus (AMF) have gained important due to their biotic role in plant growth promotes under drought stress conditions. Among mechanisms, stimulating plant growth by PGPF, production of phytohormones [7], decomposing organic matter [8], solubilization of unavailable soil bound nutrient elements [9], and protection of plants from biotic and abiotic stresses [7]. Indirect growth activators by plant growth promoter's fungi occur via niche exclusion, antibiosis, predation and mycoparasitism [10] [11]. Sometimes more than one mechanism is used to enhance growth [10]. Arbuscular mycorrhiza forming fungi (AMF) are obligate biotrophs that require the host plant to complete their life cycle. The fungus colonizes the root cortex and forms intracellular structures called arbuscules where the exchange of nutrients between the partners takes place. The extracellular hyphae spread widely into the surrounding soil, thereby reaching nutrient and improving plant growth. The role of AMF in growth promotion and stress suppression in plants is reported since the very old times [12]. The ability of AMF to promote plant growth is due to nutrient uptake, particularly phosphorus (P) [13] [14], AMF-colonized crop shows increased growth and yield [15] [16]. Xu et al. (2018) [17] showed that maize plants appeared to have high dependency on AMF which improved physiological mechanisms by raising growth, chlorophyll content, gas exchange and rubisco activity under salinity stress. Mathur et al. (2018) [18] showed that AFM plants increased relative water content both of plants leaf and soil indicating that AMF hyphae penetrated deep into soil and provided moisture to the plants. Thus this work was conducting to study the drought tolerance of maize plant at two different harvesting stages (30-and 90-days) and the combined action with mycorrhiza inoculation at these plant growth stages.

Experimental Sites and Drought Stress Treatments
Maize seeds (Zea mays L. cv. 215) were obtained from Agronomy Department, Faculty of Agricultural, Minia University, El-Minia, Egypt. Maize crop is one of the food crops that have several uses, whether as a food for man or as animal feed, due to its high nutrition value. Also, maize enters in the process of manufacturing some important products such as corn oil, fructose and starch [19].
Maize seeds were surface sterilized by immersion in a mixture of ethanol 96%

Drought Stress and Treatments with Mycorrhiza Colonization
The seedlings were left to grow under the desired soil moisture content levels (100%, 70%, 50% and 20%) in the first group of experiment, this considered untreated with inoculum (−AM

Laboratory Analysis for Metabolities
At the end of the experimental period (30-days and 90-days of harvesting) plant fresh and dry matter yield of the different organs (shoot and root) were determined. Determination of the dry matter involved harvesting and careful separation of fresh organs. Fresh organs were then dried in an oven at 80˚C. Dry matter was determined after drying plants in an aerated oven at 70˚C to constant mass. Soluble sugar was determined by the anthrone-sulfuric acids method [22].

Mycorrhiza Colonization Measurement
After 90-days from sowing, roots were carefully washed from adhering using tap water. A sample of approximately 0.5 g fresh roots from each pot was removed to estimate the degree of infection of AM using direct measurement of the amount of the fungal hyphae by measuring the total chitin after conversion to

Statistical Analysis
The experimental data were subjected to the one way analysis of variances (ANOVA test) using the SPSS version 11.0 to quantify and evaluate the source of variation and the means were separated by the least significant differences, L. S. D. at P level of 0.05% (Steel and Torrie, 1960). Experimental data were subjected to one way analysis of variance and the means were separated by the least significant differences, L. S. D. [31]. Correlation coefficients were calculated using statgraphics 5.0 software.

Results
Fresh and dry matter were decreased as decreasing moisture content in shoot and root at both 30 and 90 days of plant harvesting especially at lower moisture content levels (30% M. C.) ( Table 1). This decreasing effect was more prominent  as decreasing M. C. However, the plant harvesting after 90-days soluble sugar showed irregular decreasing effect as decreasing M. C. levels, the percent of decrease was 27.8% and 29.1% in shoot and root respectively compared with control plants (Table 2). Soluble protein was markedly lowered in shoot and root at both harvesting stages (30 and 90-days). The percent of reduction was 35.2%, 22%, 37.6%, 31.6% in shoot and root at both 30 and 90-days of plant harvesting respectively (Table 2). Total sugar showed a marked reduction in shoot and root of both harvesting stages reached a low level at 20% M. C. level (Figure 2(a)). Exit from previous trend total sugar in shoot of plant harvesting after 30-days a significantly accumulated as decreasing M. C. level (Figure 2(a)). Total protein was markedly increased in shoot of plant harvesting at 30-days while run around control value in root (Figure 2(b)). Whereas total protein tended to decrease in both shoot and root of plant harvesting at 90-days. Amino acids content run around control value 100% in shoot and root of maize plants harvesting at 30-days, while a smooth reduction was exhibited in plants harvesting at 90-days (Table 3). Decreasing moisture content induced in most cases unchanged effect in proline content in shoot at both tested harvesting stages (Table 3). In root decreasing M. C. exhibited a marked increase in proline content at both plant harvesting stages (Table 3). Potassium content was mostly increased in both tested organs of maize plants at both stages of plant harvesting (30 and 90-days) (Figure 3(a)). This increasing effect was highly recorded in shoot than in root. i.e. root is higher in increasing K + content than shoot organ. Ca 2+ and Mg 2+ content were significantly increased in shoot and root at 30 and 90-days harvesting stages (Figure 3(b), Figure 3(c)). The percent of increasing at 20% M. C. level was 114.3%, 106.3%, 128.6%, 107.7% in case of Ca 2+ in shoot and root of two harvesting stages. Also, the percent of enhancement in Mg 2+ content at that level was 109.1%, 164.3%, 200%, 133.3% in shoot and root at both tested plant growth stages, i.e. the percent of activation was highly effective in root than in shoot at 30-harvesting stage while this effect was recorded in shoot than in root at 90-days of plant growth (Figure 3(b)). Osmotic pressure in maize plant harvesting after 30-days was markedly increased up to 50% M. C. level, after that a reduction was exhibited in shoot organ while in root organ it run at irregular

Mycorrhiza Inoculation
Plant mycorrhization has resulted a highly significant increase in fresh, dry matter and water content of shoot and root compared with un-inoculated plants at both harvesting stages (Table 1). Plant inoculated with mycorrhiza in most cases accumulated soluble sugar and soluble protein in both tested organs compared with either control value or with corresponding moisture content level, this activation was more obvious at 30-days harvesting plants (  (Table 3).  Table 4 represents the degree of root colonization by AM as measured by

Discussion
From previous data it can be demonstrated that drought stress tolerant varied in different organs of the same plants and also varied among different stages of plant development. While root organ tolerate drought stress up to 20% moisture content, shoot organ exhibited a degree of sensitivity especially at 50% and 20% moisture content levels in plant harvesting after 30-days. Whereas plant harvesting after 90-days tolerate up to 70% moisture content level, after that a dramatic sensitivity was recorded presented in production of fresh, dry matter and water content. The sensitivity of maize plants was related with reduction of root soluble sugar, shoot and root soluble protein at 30-days of plant harvesting stage, and soluble sugar and soluble protein in both organs at both harvesting stages. This related with reduction in OP and lowering of water uptake which induced a marked decrease in fresh and dry matter production in shoot and root at both harvesting stages. The soluble sugar in shoot of plant harvesting at 30-days concomitant with stable value of amino acids which concomitant with increasing effect in OP and this functioning in plant survival at this stage. Also, minerals has a role in previous trend increasing K + , Ca 2+ , Mg 2+ in shoot and root in plant harvesting at 30-days and 90-days. These factors related with increasing OP value especially in shoot of both harvesting stages (30-days and 90-days). The inhibitory effect of drought on growth parameters could be attributed to the osmotic effect of water stress [32] [33]. Also, the reduction of yield may be ascribed to the harmful effect of soil moisture stress and nutrient balance disorder in root media [34], or reduced rate of new cell production may be make additional contribution to the inhibition of growth [35]. The reduction in growth criteria due to drought stress might be related to disturbance of water flow from root to shoot [36], decrease in water potential of cell sap [37], or inhibition of cell division [38]. One distinctive feature of most plants growing in stress environments is the accumulation of proline [39] and it has been inferred that there may be a relationship between cellular proline level and cell turgidity via osmotic adjustment [40] [41]. Osmotic adjustment helps to maintain cell turgor, which can allow cell enlargement and plant growth during water stress; and it can allow stomata to remain at least partially open and CO 2 assimilation to continue at water potentials that would be otherwise inhibitory [42]. Supported the previous view that drought stress is among the factors most limiting to plant productivity [43] [44]. The plant tolerance at 70% M. C. level represented in production of fresh and dry matter at 30-days of plant harvesting was parallel with increasing trend in soluble sugar and amino acids in shoot and root and root protein, shoot K + , Mg 2+ and P in shoot and root which resulted activation trend in Op in both organs at that level [45] [46] [47]. Proline content showed a variable trend while run around control value 100% in shoot, in root it was significantly increased as decreasing moisture content at both tested harvesting stages. This increasing effect of drought stress in proline content in root organ can be consider as a sign of tolerant as decreasing M. C. levels of plant harvesting at 30-days.While it consider as a sign of sensitivity in root of plant harvesting at 90-days. i.e. the causes of proline accumulation were varied at different stages of plant growth. Lutts et al., (1999) [48] suggested that proline accumulation in rice under water deficit was most likely a symptom of injury rather than an indicator of increased tolerance. This activation in OP with AM reflected in increasing water uptake under different level moisture content in shoot and root of both harvesting stages. Also enhancement effect on soluble sugar, soluble protein and amino acids was shared in OP effect. This induced an increasing effect on photosynthetic efficiency which in turn permits increasing effect in carbohydrate and nitrogen metabolism. These reflected on increasing maize drought tolerance as presented in growth yield [53]. Osmolytic accumulation in plant cells can act as a mechanism of osmotic adjustment for decreasing the cellular osmotic potential and thus for maintaining water absorption and turgor. Osmolytic accumulation can also protect cellular components, such as cell membranes and proteins, and sustain the physiological activity of plants [54]. Proline content with mycorrhiza inoculation was more or less unchanged in shoot of plant harvesting at 30-days and in root of plant harvesting at 90-days. However, a marked increase was induced in plant harvesting at 30-days and in shoot of plant harvesting at 90-days. This indicated that proline was response varied in different organs and in different harvesting stages. It can be consider in these organs which accumulated as a sign of osmotic adjustment [41]. The colonization of roots by AM fungi in various plant species induces proline accumulation when water is limiting [55]. The enhanced accumulation of proline in these studies was linked to AM-induced drought resistance with proline acting as osmoprotectant. Conversely, in several studies, while proline content increased in response to water deficit, a lower accumulation of proline has been observed in mycorrhiza plants relative to nonmycorrhizal counterparts. The enhanced accumulation of proline in these studies was linked to AM-induced drought resistance with proline acting as osmoprotectant. Conversely, in several studies, while proline content increased in response to water deficit, a lower accumulation of proline has been observed in mycorrhiza plants  [61] showed that lower proline accumulation in AMF plants under drought stress.
Our results therefore suggest that AMF strongly altered leaf sucrose and proline metabolism through regulating sucrose-and proline-metabolized enzyme activities, which is important for osmotic adjustment of the host plant. Also AM significantly accumulated total sugar and total protein which served in increase plant efficiency to increase dry matter of both testing harvesting stages. Chun et al. (2018) [62] reviewed comprehensively compiles significant correlations and limitations associated with plant stress tolerance and evasion mechanisms. Proline has every possibility of consideration as an indicator and potential marker for possible injury by osmotic stress.Wu and Ning Zou (2017) [63] have studies indicated a quick response to, drought and salinity stresses involving several mechanisms, such as root morphological modification, reactive oxygen species change, osmotic adjustment, direct absorption of water by extra radical hyphae, up-regulated expression of relevant stressed genes, glomalin-related soil protein release, etc. The underlying complex, multi-dimensional strategy is involved in morphological, physiological, biochemical, and molecular processes. The AMF responses are often associated with homeostatic regulation of the internal and external environment, and are therefore critical for plant health, survival and restoration in native ecosystems and good soil structure. The present work showed that AM infection levels measured by N-acetyl glucosamine content were not affected by drought stress. However these results are consistent with those Simpson and Dafit (1990) [64] [65] and Smith and Read (2008) reported that AM infection levels of maize and sorghum were not affected by water stress. Results showed also that control roots contained N-acetyl glucosamine would be attributed to mycorrhiza and other fungi naturally present in soil. This study indicates that improvement of maize grown under different moisture content by mycorrhiza inoculation could be attributed to improved water and mineral up-take especially K + , Ca 2+ , Mg 2+ rather than P. In contrast with some published studies [66] [67]. Mychorrhiza symbiosis mitigated the accumulation of total sugar and total protein which served in increasing dry matter of shoot and root of two tested harvesting stages.

Conclusions
From previous results, it can be concluded that: 1) Drought tolerance varied in different organs and also at different stages of harvesting.
2) Increasing soluble sugar, soluble protein, amino acids, K + , Ca 2+ , Mg 2+ and P with AM inoculation can be served in increasing OP values, increasing water uptake and hence growth yield.
3) AM application induced an accumulation of total sugar and total protein which served in increasing dry matter of shoot and root of two tested harvesting stages.
4) Proline can be record as a sign of osmotic stress injury as in root of plant harvesting at 30-days and 90-days or as contribution in osmotic adjustment as in shoot of both harvesting stages under decreasing moisture content.