Effect of Saline Irrigation Water, Gibberellic Acid(GA3) and Biofertilizers on Growth, Flowers Yield and Oil Production of Matricaria Chamomilla, L.plants

INTRODUCTION

Chamomile (Asteraceae; Matricaria Chamomilla, L.) is considered one of the oldest, most widely used and well documented medicinal plants in the world  The original habitat of chamomile species are Northern Africa, Asia, Southern and Eastern Europe. Chamomile is an annual plant. It has a thin spindle -shaped roots only penetrating flatly into the soil. The branched stem is upright , heavily ramified, and grows to a height of 10–80 cm. The long and narrow leaves are bi- to tripinnate. The flower heads are placed separately, they have a diameter of 10-30 mm, and they are pedunculate and heterogamous. The golden yellow tubular florets with 5 teeth are 1.5-2.5 mm long, It always ends in a glandulous tube. The 11-27 white plant florets are wide (3.5) mm, long 6-11 mm, and arranged concentrically. The receptacle is 6-8 mm wide, flat in the beginning and conical, cone-shaped later, hollow-the latter being a very important distinctive feature of Matricaria chamomilla - and without paleae. The fruit is a yellowish brown achene (Franz et al., 2005).

Chamomile is a well-known medicinal plant in folk medicine cultivated all over the world. Chamomile essential oil is widely used in pharmaceutics, cosmetic, and food industries. The biologically active substances in Chamomile essential oil are trans -β-farnesene, α-bisabolol, bisabolol oxides, chamazulene, and enyn-diccycloethers ( Brunke et al., 1992 and Grgesina et al.,1995). The flowers heads are the main organs of the production of essential oil, It may exceed 120 chemical constituents in chamomile flower  as secondary metabolites , including 28 terpenoids, 36 flavonoids, and 52 additional compounds (Mann and Staba, 2002).

        Saline irrigation water is considered detrimental to most crop plants. Even salt-resistant plants show decrease in yield when grown under saline irrigation. However, saline water is available in abundance in many countries of the world, thus, the importance of establishing agro-management regimes that include saline water is self-evident . In arid and semi arid regions, salinity has been recognized as an important factor influencing crop production and agricultural sustainability. On the other hand, irrigation with saline water without proper management, such as mixing with fresh water, would produce adverse effects on crop yield and soil productivity due to deterioration of soil quality.(Afifi et al., 1998), Morsy (2003).

Gibberellic acid is among the many plant hormones, It had an axis  the main focus of some plant scientists to relieve the negative effects of salinity (Basalah and Mohammad, 1999). Available information about the effect of GA3 on the growth of plants under salinity it may be limited and  few reports have stated the ability of GA3 to reduce the negative effects of salinity (Misratia et al., 2013).Use of The exogenous applications of gibberellic acid leads to some benefit in relieve the adverse effects of salt stress and also improves growth, development  and yield quality and seed yields (Javid et al., 2011). It was reported that foliar application of GA3  improved chlorophyll content and water use efficiency (Misratia et al., 2013).

            Bio-fertilizers are reasonably safer to the environment and play an important role in decreasing the use of chemical fertilizers. Consequently, it cause a reduction in environmental pollution. Bio fertilizers are microbial inoculants consisting of living cells of micro-organism like bacteria, algae and fungi alone or combination which may help in increasing crop productivity. Bio fertilizers can influence plant growth directly through the production of phytohormones such as gibberellins, cytokinins and IAA that act as growth regulators and indirectly through nitrogen fixation and production of bio-control agents against soil-borne phytopathogens and consequently increase formation of metabolites which encourage the plant vegetative growth and enhance the meristematic activity of tissues to produce more growth (Ahmed and Kibret, 2014).

The objectives of the present study were to investigate the effect of the salinity with different gibberellic acid concentrations, biofertilizers  applications and their interactions on the growth , flowering parameters and quantity of the essential oil of Matricaria Chamomilla, L. plants.

MATERIALS AND METHODS

The pot experimental study was carried out during two successive seasons of 2015 /2016 and 2016/2017 at the Nursery, Department of Floriculture, Ornamental Horticulture and Landscape Gardening, Faculty of Agriculture, Alexandria University, Egypt.

The plant used in this experiment was chamomile (Matricaria Chamomilla L. var." German chamomile ").

In the first season 2015/2016, Seedlings were obtained used in the experiment, uniformly shaped seedlings from a commercial nursery in (Kerdasa) in Cairo (planted its seeds on 2nd October 2015)Which reached, the average seedling length is 15 cm(one seedling per pot and an average of 15-20 leaves per seedling ) were transplanted to the final pot, 30 cm diameter filled with clay, sand and cattle manure (2:1:1) on 3rd  December 2015 in full sunny place . In the second season 2016/2017, The seeds resulting from the first season were planted on 4th October 2016, and seedlings were selected from them with the same specifications as the seedlings of the first season. They were planted according to the standards of the first season in the final pot on December 6th, 2016 respectively. Chemical properties of the growing media used in the study are presentedin Table 1 .

Table(1)Some Physical and chemical analysis of the chosen growing media according to Jackson ( 1973 )

Experimental treatments and layout:

The experimental treatments were consisted of three concentrations of saline water Sodium chloride i.e. (tap water as control) 0.56 dSm-1, 3 dSm-1 and 6 dSm-1  used. Salinity levels were obtained by addition of appropriate amount of dry NaCl to water. (Saline irrigation water started after 14 days from planting of the seedlings, alternatively irrigation every 3 days with saline and tap water for two months and three levels of gibberillic acid  (GA3) at  0, 100 and 200 mg L-1 were sprayed three times after 30, 45, 60 days from the transplanting. The biofertilizer was used either nitrobein contained nitrogen fixing-bacteria (Azotobacter and Azospirillum) and phosphorein (a biofertilizer contains a specific clone of bacteria which changes the unavailable triphosphate to available monophosphate ) were mixed with the surface layer of the soil It was added two times, the first was immediately added at direct transplanting and the second was added after one month later B0 (without biofertilizer), B1 (5 g nitrobein+ 5 g phosphorein) and B2 (10 g nitrobein+10 g phosphorein) per plant.

 Experimental layout and statistcal analysis

The experiment layout was designed to provide a split-split plot experimental design  which containing three replicates, each replicate contained (27 treatments).  Three (3) pots were used as a plot for each treatment (Snedecor and Cochran, 1981).

The whole plots were represented by three Salinity treatments. The sub plots were randomly assigned the three gibberillic acid treatment  (GA3) levels and the sub-sub plots were described the three treatments of biofertilizers.

Morphological Measurements:

1-    Vegetative growth characteristics: Plant height (cm), plant total fresh weight (g),   total dry  weight (g) Conducted 70 days after transplanting .

2-    Yield parameters : Number of flower heads per plant, fresh weight  of  flowers yield (g), dry weight  of  flowers yield (g) (The beginning of collecting inflorescences with the end of December and the beginning of January).

Essential Oil

The oil percentage was determined by water distillation method according to the (British Pharmacopoeia, 1963), where the flower from each sample (50 g fresh flower) were placed in the flask of two liters capacity (the amount of water and sample was 2/3 from flask ). A proper essential oil trap and condenser were attached to the flask. The distillation was continued for 5 - 7 hours until no further increase in the oil was observed.

1-Essential oil percentage (%)

Essential oil % = Oil volume (reading measured pipette) / weight of sample (g) ×100.

2-Essential oil yield

Oil yield/plant= Oil percentage × fresh weight of flower heads per plant.

The same steps and techniques of the first experimental year 2015/2016 were followed in the second one 2016/2017.

The GC analysis of the volatile oil samples was carried out using gas chromatograph instrument stands at the Medicinal and Aromatic Plants Dept. Laboratory, Horticulture Research Institute (Agricultural Research Center,Cairo, Egypt,  Ministry of Agriculture and land Reclamation)

The data were statistically analyzed according to the methods described by Snedecor and Cochran, (1981) using L.S.D. to compare between means of the treatments.

Results and Discussion

          Results presented in Table  (2) showed that height of  chamomile plants was significantly affected by irrigation water salinity, gibberillic acid  concentrations, biofertilizer applications and their interactions in seasons 2016 and 2017, except the interaction between the  salinity levels and GA₃ concentration in the second seasons.

Plant height of chamomile  gradually decreased with increasing salinity level, where the tallest plants of 41.45 and 54.55 cm in the first  and second seasons, respectively, resulted  by using tap water, however the  shortest  plants of 39.24 and 50.76 cm in the two successive seasons resulted by  using saline irrigation water ( EC, 6 dSm^-1). The  decrease in  plant  height resulted  from soil  salinity might be attributed to the inhibition of both meristematic activity and elongation of  cells (Neiman,1965). Tuna et al.(2008) also reported that decreasing plant height with increasing salinity levels resulting to inadequate  water uptake and hence, relative water  content (RWC) was significantly  decreased and resulted in limited water availability for  the  cell extension process . Similar results were obtained by many researches as Ali and Hassan (2012) on Matricaria chamomilla, . GA₃ application at 100 or 200 mg L^-1 significantly increased plant height by 15.79 and 13.38% respectively, in the first season and 8.29 and 8.69% in the second season compared with control, however the  difference between 100 and 200 mg L^-1 did  not reach significantly level. These results might be due to the promotive effect of gibberillins on  increasing the auxin level of tissue or cause  cell division and cell elongation (Kuraishi and Muri,1964). Similar results were reported  by Saedi and Al-Rubaiee(2012) on Matricaria chamomilla, and Atteya and El-Gendy(2018) on Tagetes patula.

Data in the previous Table, also showed that  increasing biofertilizers gradually increased chamomile plant height and the maximum heights  were 40.90 and 53.33 cm in the two successive seasons resulted from 10g nitrobein + 10g phosphorein application. The positive effect of  biofertilizers are in line with those obtained by Gewaily et al.(2006) who  reported that biofertilizer

application enhanced the  vegetative growth and  plant  height of  Majorana  hortensis L. Similar results were reported by El-Naggar  et al. (2015) on Ocimum basilicum .

Concerning the three factors interaction effect on chamomile plant height, data in Table (2) demonstrated that the  tallest plants in the two seasons, generally resulted from irrigation with  unsaline water  combined with 100 or 200 mg L^-1 of gibberillic acid and  biofertilizers (5 g or 10 g nitrobein +5 g or 10 g phosphorein) per pot. These results could be due to the positive  integrated  effect of gibberillic acid and  biofertilizer  to alleviate  the  stress of salinity on  plant  growth, where Azospirillum  brasilense produced  plant  hormones, especially growth promoters and supply plants with combined nitrogen. These results agreed with those reported by Ali and Hassan (2012) on Matricaria chamomilla.

Table (2):Effect of water salinity, GA3 , biofertilizer and their interactions on plant height (cm) of Matricaria chamomilla L. plants in the seasons of 2016 and 2017.

1.2.Total fresh weight (g) per plant.

 Total fresh weight of chamomile plant  significantly affected by salinity levels, gibberillic  acid concentrations,  biofertilization application rates and their  interactions in seasons of 2016 and 2017, except GA3 combined with biofertilizer levels  and GA3 x salinity levels in the first and second seasons, respectively (Table 3).

Increasing salinity levels to 6 dSm-1 significantly decreased chamomile plant fresh weight by 2.24 and 5.34 (g) in 2016 and 2017 seasons, respectively. In this respect, The decrease in fresh weight might be due to the high level of salinity which increased osmotic pressure and caused a drop in plant water content and inhibition of both meristamatic activity and elongation of cells ,or at might return  the reduction in weight to inhibition of the biosynthesis foods and their translocation to the growing shoots . These results are in general  agreement with  those reported by Heidari  and Sarani(2012) on Matricaria chamomilla and Estaji et al.(2018) on Satureja hortensis.

Spraying chamomile plants with gibberillic acid  up to 200 mg L-1significantly increased  plant fresh weight, however the difference between 100 and 200 mg L-1 was insignificant in the two seasons of study. the increases were 4.57 and 8.33 % and 9.31 and 14.51 % as increasing  GA3 concentrations up to 200  mg L-1 in the first and second  seasons, respectively. The  promotive  effect of gibberillins on growth may be  increasing  the auxin level  of tissue or enhance the conversion of tryptophan to IAA,  which  cause cell division and cell elongation .Similar results were obtained by Reda et al.(2010) on Chamomile recutita.

Concerning  biofertilizer  application  effect on that trait, presented results  in Table (3)  revealed that 10 g nitrobein + 10 g phosphorein per plant  produced  the highest plant fresh weight 35.54 and 45.82 g in the two successive seasons,  however the difference between (5 g nitrobien  +  5 g phoshorein) and control did not reach  the significance level. The positive effect of biofertilizer might  be due to  promote plant growth and  phytohormones and bio-control agents production against  soil- borne  phytopathogens (Cohen et al.,2007). The  present results are in general agreement  with those obtained by Shalan et al.(2001) on chamomile, and Mahfouz et al. (2003) on Majorana hortensis moench.

With respect to  salinity levels with  gibberillic  acid concentrations combined with  biofertilizer  levels interaction effect  on chamomile plant   fresh weigh, results presented in Table (3) showed that the highest plant  fresh  weights 44.0 and 37.79 g  resulted  from  irrigation with  saline  water( EC, 3 dSm-1), fertilized  with  10 g nitrobein  + 10 g phosporein and spraying with  100 and 200 mg L-1GA3 concentrations. Also, using saline water ( EC, 6 dSm-1) and 10 g nitrobein  + 10 g phosphorein per plant  without  gibberillic acid application, however, using  tap water,  generally produced the highest plant fresh weight and GA3 and biofertilizer application in the first season. 

Fertilized chamomile plants  with  5 g nitrobein  + 5 g phosphorein    combined  with 200 mg L-1giberrillic produced the highest plant fresh  weight 49.95 and 50.30 g using  tap water and saline  water (6 dsm-1), respectively, in the second season.  Under ( EC, 3 dSm-1) salinity level, 10 g nitrobein  + 10 g phosphorein  without GA3 application or 200 mg L-1, also  produced the highest plant fresh weight 50.21 and 52.33 g  , while  using  5 g nitrobein  + 5 g phosphorein   and spraying  100 and 200 mg L-1GA3 under  the same salinity level, also produced the highest fresh  weight per chamomile plant 53.45 and 56.05 g , respectively, as shown in Table (3). Improvement  of   chamomile plants  growth by gibberillic acid application could result in an enlargement of leaf area, the motivation of cell division  and / or cell elongation, simulation of photosynthetic rate, modified partitioning of photosynthates, or in their combinations. Also GA3 accumulates of hexoses which considered  important for the primary cell wall biosynthesis, accordingly enhancing  plant growth  under saline  stress conditions (Khan et al., 2015). On the other hand, Morteza et al. (2013) on Lippia citriodors,  recorded positive effects  of  biofertilizers on  plant growth  as a result of increasing photosynthesis  tissuse by promoting  the absorption  of nitrogen and phosphorus  that have effects chlorophyll production and plant essential enzymes preparing.

Table (3):Effect of water salinity, GA3 , biofertilizer and their interactions on total fresh weight / plant(g) of Matricaria chamomilla L. plants in the seasons of  2016 and 2017.

1.3. Total dry weight /plant (g).

Total dry weight of chamomile plant  as presented in Table (4)  was significantly affected by salinity levels, gibberillic acid concentrations andbiofertilizer rates besides their  interactions  in the two studied seasons.

Irrigated chamomile plants  with  tap water (unsaline water) produced the highest plant dry weights of 11.72 and 15.28 g,  in 2016 and 2017 growing seasons, respectively. Conversely, saline water ( EC, 3 and 6  dSm-1) in the first season  and (EC, 3dSm-1 ) in the second season produced the lowest plant dry weights (10.51,10.50 and 13.68 g), respectively. In this respect, decreasing fresh and dry weight of herb with increasing salinity levels may be due to the decrease in the growth resulting from the inhibition of photosynthesis that reduced carbohydrates storage(Ahmed et al.,2011). These results agree with those obtained by Deepika et al.(2015)onMatricaria chamomilla, and Estaji et al.(2018) on Satureja hortensis.

Results presented in that Table, also, indicated that the highest  total  dry weights of chamomile plants  11.90,15.02 and 16.14 g resulted from spraying gibberillic acid by 200 mg L-1 in 2016 and 100 and 200 mg L-1in season 2017, respectively. This increase might be due to  the promotive effect of  gibberillins on plant growth on increasing  the level  of auxin of tissues  or  enhance the conversion of  tryptophan to IAA, which cause cell division and cell elongation (Kurashi and Muir, 1964). Increasing biofertilizer  levels gradually increased  plant total dry weight and  10 g nitrobein + 10 g phosphorein gave  the highest plant dry weight of 11.54 and 15.23 gfollowed by 5 g nitrobein + 5 g phosphorein (10.78 and 14.49 g in the first  and second seasons, respectively). That could be attributed  to effect of  biofertilizers on plant hormones creation, improving  phosphorus  solubilization  and  nutrient mobilization and hence   increasing plant growth (Rashed et al.,2017) on Nigella sativa. The present results are in agreement with those obtained by Eid and El-Gawwas(2002) on marjoram, Hassan et al.(2015)on Rosmarinus officinalis.

Considering the second  order  interaction effect, results presented in Table (3)  demonstrated that  gibberillic acid and biofertilizers  application, generally increased plant total dry weight , especially with increasing  salinity levels up to 6  dSm-1 in the two seasons. That might  be due to  gibberillic acid effect on cell enlargement under  salt stress (Achard et al.,2006), however   biofertilizer promote salinity tolerance by enhancing  nutrient acquisition and maintenance of the K+ : Na+ ratio (Smith and Read,2008) and altering the hormonal profiles (Aroca et al.,2013). The  present results are in agreement with  those  were obtained by Ali and Hassan (2012) on Matricaria chamomilla and Rashed et al.(2017) on Nigella sativa.

Table (4):Effect of water salinity, GA₃ , biofertilizer and their interactions on total  dry weight / plant(g) of Matricaria chamomilla L. plants in the seasons of  2016 and 2017.

2.Flower yield characteristics:

2.1. Number of flower heads (inflorescences) per plant.

Results presented in Table (5)  revealed that number of flower heads per chamomile was significantly affected by the three studied factors, i.e. salinity levels of   irrigation water, gibberillic acid  concentrations and biofertilizer  rates besides all the two and three factors interactions  in the two seasons. Increasing salinity levels to 6  dSm-1 gradually decreased the number of flower heads by 80.03 and 137.73 heds / plant in the first  and second seasons, respectively, that could be due to the adverse effect of salinity on number of branches/ plant (Ali and Hassan, 2012) on Matricaria chamomilla . These results agreed with those  reported by Kamkari et al.(2016) on pot marigold. With regard to GA3 effect, obtained results showed that  increasing concentration up to 100 mg L-1produced the highest number of flower heads/ plant 444.51 - 504.69 in seasons 2016 and 2017, respectively. These  results agree with  those were reported by Amiri et al.(2014) on Matricaria recutita and Palei et al. (2016) on African marigold. Biofertilizer application significantly increased  by 19.16 and 33.66 heads with  5 g nitrobein + 5 g phosphorein  and 10 g nitrobein + 10 g phosphorein  in the first season and 97.65 and 59.97 heads, respectively, in the  second  season over than the control . Positive  effect of biofertilizer could  be due  to plant  hormone creation , phosphorus  solubilization  and nutrient  mobilization (Rashed et al., 2017) on Nigella sativa.  These findings  are in agreement  with  those of Mashhadi et al. (2017) on chamomile. Concerning the three factors interaction effect on number of flower  heads/ plant,  results presented in Table (5) showed   that the  highest  number of flower  heads 614.83 and 629.67 resulted  from  10 g nitrobein + 10 g phosphorein   biofertilizer application without  or with 100 mg L-1 GA3 under irrigation with  tap water, respectively, besides 100 mg L-1 gibberillic acid  combined  with  5 g nitrobein + 5 g phosphorein  / pot under irrigation with  saline water (EC, 6 dsm-1). However, the highest  number of flower heads /plant under irrigation  with  tap  water and  10 g nitrobein + 10 g phosphorein  /pot 605.10 and 648.29 at 0 and 100 mg L-1 GA3, respectively, and 669.65 and 616.94 flower  under 5 g nitrobein + 5 g phosphorein  and 100 and 200 mg L-1GA3, respectively. Besides 622.64 and 708.41 flower heads that resulted under  saline water (EC, 3 dsm-1), fertilization with 5 g nitrobein + 5 g phosporein  with 100 mg L-1  GA3. These  results clearly  showed  the biofertilizers and  GA3 effect on nutrient mobilization, hormone creation. Also, the development flower primordial was  greatly, influenced by  growth regulators (Kumar et al, 2010).

Table (5) Effect of water salinity, GA3 , biofertilizer and their interactions on number of flowers head  / plant of Matricaria chamomilla L. plants in the seasons of  2016 and 2017.

2.2.Fresh weight  of  flower heads yield (g/plant).

Results  presented in Table  (6)  revealed that fresh weight  of flower heads yield   per chamomile plant was significantly affected  by salinity levels of irrigation water, GA3 and biofertilizer application  besides all  second and three  factors interactions in 2016 and 2017 growing seasons. Increasing salinity stress up to EC, 3 and 6  dSm-1 led to significantly  decreased in fresh weight of flower heads yield / plant by 20.90 and  28.40 %, respectively, compared with irrigation with nonsaline water in the first season. However, these percentages  were 14.59 and 27.64 % , respectively, in the second season. These reactions could be  due to the adverse effects  of salinity stress on apical growth of chamomile plants and endogenous hormonal imbalance, besides  sometimes it could  be due  to the lethal effects of  Na+ and Cl-  ions or adverse water relations (Younis et al.,2010).These  results are in agreement  with  those of  Kamkari et al.(2016) on pot marigold. Concerning  gibberillic  acid   effect, the  results indicated  that  100 mg L-1 GA3 produced the highest  flower heads fresh weight per chamomile  plant(42.60 and 47.57 g/ plant) in  seasons of 2016 and 2017, respectively, with  insignificant  difference  without GA3 in the first season. Conversely,  the highest GA3 concentration (200 mg L-1) produced  the lowest flower heads yields of 37.01 and 36.08 g/ plant in the first and second seasons, respectively, that might be due to gibberillic acid stimulation effect  on the vegetative growth, mainly (Khan et al. ,2015).These results are in harmony with  results of Reda et al. (2010) on Chamomile recutita. .

Results in Table (6), also revealed that fresh weight of flower heads yield per chamomile plant enhanced progressively with  increasing biofertilizer levels from 0 to 10 g nitrobein + 10 g phosphorein  per pot . The maximum fresh flowers yield / plant was 45.48 and 47.71 g in the first and second seasons, respectively,  resulted  from  10 g nitrobein + 10 g phosphorein / pot. That might  be due to that biofertilizers enhanced the  vegetative  growth and consequently number of branches / plant  will  increased (Cohen et al. ,2007).These  results are in line with  those  reported by Ali (2001) on Calendula officinallis and Mashhadi et al.(2017) on chamomile . With respect to the  second order interaction effect on that  trait, obtained results pointed  out that irrigation chamomile   with nonsaline water and  high biofertilizer level(10 g nitrobein + 10 g phosporein  /pot) without giberrillic acid spraying or spraying  GA3with 100 mg L-1 produced  the highest   fresh flower heads yield  per plant 70.36 and 69.25 g, respectively, in the first season and 76.78 and72.75g. in the second season.

Table (6):Effect of water salinity, GA3 , biofertilizer and their interactions on total fresh weight of flowers head  yield (g/plant) of Matricaria chamomilla L. plants in the seasons of  2016 and 2017.

2.3.Dry weight  of  flower head (yield)(g /plant).

Presented data in Table (7) indicated that salinity levels of irrigation water, gibberillic acid concentrations, biofertilizer application levels and their interactions between  the three studied  factors had significant effects on dry weight of flower head yield/plant  in the two growing seasons. Irrigated chamomile plants with fresh water produced significantly highest dry weight of flower head yields 17.37 and 18.22 g/plant in the first and second seasons, respectively. On the other hand, increasing salinity levels of irrigations  water up to EC, 3 and 6  dsm-1  significantly  decreased dry weight of flower head  yields per plant by 29.19 and 35.0%, respectively, in the first season and 33.10 and 37.05%  in the second season, but the differences between the two salinity levels  did not  reach significance level. These  might be due  to the adverse  effects of  saline stress on apical growth of  chamomile  plant and   imbalance  of  endogenous  hormones (Younis et al.,2010).Similar results were obtained by Dadkhah(2010) on Matricaria chamomilla, Mogahdam et al.(2014)on Matricaria recutita, Kamkari et al.(2016) on pot marigold. With regard to giberrillic acid concentrations effects, results showed that  100 mg L-1application produced the highest dry weight of flower head yield 13.84 and 15.62 g/plant in seasons of 2016 and 2017, respectively, however this GA3 concentration was statistically equaled without  GA3 spraying 14.45 g/plant in the  first season. It may be because GA3 in salt stressed plants showed an increase in photosynthetic capacitya vital factor for higher dry matter synthesis ( Misratia et al.2013).These findings agree with  those reported by Reda et al. (2010) on Chamomile recutita and Palei et al. (2016) on African marigold. With  respect to biofertilization effect, results presented  in  Table (7) cleared that the highest dry weight  of flower heads yield was 14.67 and 15.15 g/plant resulted  from  application the highest  biofertilizer  rate (10 g nitrobein + 10 g phosphorein / pot) in the first and second seasons, respectively. That might be due the enhancement effect of  biofertilizers on  vegetative growth and consequently  increased number of branches per plant (Cohen et al.,2007).These results agree with those reported by Ali (2001) on Calendula officinallis, Mashhadi et al.(2017) and Mostafa et al.(2019) on chamomile.

Considering the interaction between the three  studied factors on dry weight  of flower heads yield  per chamomile plant in the two growing seasons, obtained results indicated  that irrigation of chamomile  plants with fresh  water, spraying with 100 mg L-1giberrillic acid and biofertilized with 10 g nitrobein + 10 g phosporein / pot produced the highest dry weight of flower  heads yield of 24.03 and 25.24 g/plant in the first and second seasons, respectively, besides 5 g nitrobein + 5 g phosporein  combined with 100 mg L-1GA3 23.89 g/plant and10 g nitrobein + 10 g phosphorein without GA3 application 23.52 g/plant under irrigation with  fresh water in the second season.

Table (7):Effect of water salinity, GA3 , biofertilizer and their interactions on total dry weight of flowers head yield (g/plant) of Matricaria chamomilla L. plants in the seasons of  2016 and 2017.

3.Essential oil productivity

3 .1.Oil percentage (%).

Obtained results in Table (8) revealed that oil percentage  in chamomile flowers  significantly affected by salinity levels of irrigated water, spraying with different concentrations of gibberillic acid and biofertilizer application levels, beasides their interactions in the growing seasons.Oil percentage was positively affected  by salinity levels, where it gradually increased by 0.06 and 0.15 % in the first season and 0.05 and 0.09 % in the second season by increasing  levels of salinity to EC, 3 and 6  dSm-1 , respectively, compared  with the control treatment. This may be due a positive correlation between the stress level imposed on the cells and the percentage of oil in the tissue. The increased percentage of oil may result from altered oil biosynthesis under stress, and from restriction of leaf area expansion which can result in denser oil glands compared to the non-stressed leaf Morales et al. (1993) suggested that an increase in oil content in some of the salt stressed plants might be attributed to the decline of the primary metabolites due to the salinity effects, causing intermediary products to become available for secondary metabolites synthesis. Salt stress may also affect the essential oil accumulation indirectly through its effects on either net assimilation or the partitioning of assimilate among growth and differentiation processes .These results are in line with those were obtained by Elhindi et al. (2016) on Ocimum basilicum and Estaji et al.(2018) on Satureja hortensis.

Spraying chamomile plants with 100 and 200 mg L-1 gibberillic acid led to an increase in essential oil percentage by 0.04 and 0.11% in season of 2016 and 0.05 and 0.09% in season of 2017, respectively, compared with untreated plants. GA3 application on lavender plants increased the light efficiency and assimilation potential of plants leading to intensified secondary metabolites production and increased volatile oil biosynthesis and storage (Hassanpouraghdam et al. 2011) on Lavandula officinalis Chaix.. Also, it is likely that elevated leaves fresh and dry weight of plants under GA3 application and concomitant increase in assimilation potential led to the suitable interactions of primary and secondary metabolism in favor of essential oil production (Marshner,1995) .The same trend of results have been reported by Sharaf El- Din et al.(2009) on Melissa officinali and Kumar et al. (2012) on Mentha piperita.

Presented results, also indicated that biofertilizer application significantly increased oil  percentage in the two  studied seasons. The maximum oil percentage 0.53 and 0.56% in the first and second seasons, respectively, resulted from fertilized plants with 10 g nitrobein + 10 g phosphorein / pot. That could be due to the importance of fertilizers for increasing mineral absorbtion and chlorophyll content in plants. The effect of biofertilizer on increasing the essential oil synthesis in the herb might be attributed to their enhancing effect on increasing the uptake of nutrients by plant roots especially phosphorus element as phosphate groupone linked by (ATP).Hence,the biosynthesis of essential oil is dependent on inorganic P content in the plant. These findings are in consistent  with those were reported by many authors such as Hassan et al. (2015) on Rosmarinus officinalis and Mostafa et al.(2019) on chamomile.

Regarding the interaction,i.e. salinity levels, GA3 concentrations and biofertilization rates effect on oil percentages, obtained  results showed that the highest  percentages of oil was 0.72% in the first season and 0.68% in the second season one resulted  from  spraying 200 mg L-1giberrillic acid combined with 10 g nitrobein + 10 g phosphorein /pot under irrigation  chamomile plants with  saline water ( EC, 6 dSm-1). Conversely, irrigated chamomile plants with  fresh water combined without or with  biofertilizer application produced  the lowest  oil percentages 0.40, 0.39 and 0.41%, respectively, in season of 2016 and 0.42, 0.42 and 0.44% in season of 2017.

Table (8) Effect of water salinity, GA3 , biofertilizer and their interactions on oil  percentage (%) of Matricaria chamomilla L. plants in the seasons of  2016 and 2017.

3.2.Oil yield (ml/plant).

Data presented in Table (9) showed that essential oil yield/plant was significantly affected by three studied factors, i.e. salinity levels of water, gibberillic acid spraying and biofertilizer   application, besides their interactions in the two seasons of study.   

Increasing salinity levels to EC, 6 dSm-1 gradually decreased oil yield/plant by 0.06 and 0.10 ml in season of 2016 and 0.11 and 0.17 ml in season of 2017 as a result of increasing salinity levels up to 3 and 6 dSm-1 , respectively, compared with  fresh water. That could be due to the adverse effect of salinity on nutrient balance,cell division  and  expansion and consequently on plant growth, as well as a series of metabolic functions (Caia et al.,2014) on Rosa hybrid. The essential oil yield in aromatic plants may be affected positively or negatively by the salinity levels (Neffati et al., 2011) and also by the type and amount of fertilizers and cultivation practices applied (Chrysargyris et al., 2016). Charles et al.(1990) stated the stimulation of essential oil production under salinity could be due to a higher oil gland density and an increase in the absolute number of glands produced prior to leaf emergence. These findings are in consistent with those of Bonacina et al.(2017) on Melissa officinalis.

However, oil yield/plant was significantly increased with increasing giberrillic acid concentrations up to 200 mg L-1and maximum essential oil yield per chamomile plant 0.23 and 0.26 ml in the first and second seasons, respectively. That might be due to the role of giberrillic acid in leaf expansion and stem elongation (Magome et al.2004).Also,it increased photosynthetic capacity and consequently  produced higher growth and dry matter under salt stress. As well as, Hassanpouraghdam et al. (2011) they reported that among phytohormones and plant growth regulators (PGRS) have crucial impact on primary and secondary metabolism of plants. Essential oil production and accumulation of volatile oil bearing plants positively responds to these molecules especially their synthetic ones at external applications and there is strong evidence that, GA3 had constant effects on plants growth and development, and consequently their active principles content and yield. These results are in agreement with Ali and Hassan (2012) on Matricaria chamomilla.

Biofertilizer application to chamomile plants led to gradually increase  essential oil yield plant, where 10 g nitrobein + 10 g phosporein  produced  maximum oil yield per plant 0.24 and 0.26 ml in 2016 and 2017 growing seasons, respectively. Carg and Manchanda (2008) on Cajanus cajan, reported that biofertilizers reduce the negative effects  of salinity, improve plant growth  rate  and antioxidant enzyme activities. Also, it could be because of the production of indole acetic acid, gibberellins and some unknown determinants by plant growth-promoting rhizobacteria PGPR, results in increase in root length, root surface area and number of root tips, leading to an enhanced uptake of nutrients thereby improving plant health under stress conditions (Egamberdieva and Kucharova, 2009).These results are in agreement with Mostafa et al. (2019) on chamomile.

           Regarding the three factor interaction between salinity levels plus gibberillic acid spraying x biofertilizer  application effect on essential oil yield  per chamomile plant  in the two studied seasons, results presented  in Table(9) showed that application of 10 g nitrobein + 10 g phosphorein  per plant combined with 100 and 200 mg L-1gibberillic acid under  irrigation with fresh water produced the highest oil yield/plant 0.33 and 0.35 ml, respectively, in season of 2016 and 0.36 and 0.39 ml in season of 2017, besides 0.34 ml/plant that resulted from combination between  irrigation with fresh water  and 10 g nitrobein + 10 g phosphorein/pot in the first season. Generally, produced the lowest  oil yield per plant with or without gibberillic acid spraying  and biofertilizer application in the two seasons.

Table (9):Effect of water salinity, GA3 , biofertilizer and their interactions on oil yield /(ml/plant) of Matricaria chamomilla L. plants in the seasons of  2016 and 2017.

CONCLUSION

In the conclusion, the results obtained from the present investigation indicated  that irrigated  chamomile (Matricaria Chamomilla L.) var.(German chamomile) with tap water combined with  spraying  gibberellic acid  three times by (100 and 200 mg L-1) and biofertilizer  application  (nitrobein +  phosporein)  by 5 g or 10 g from  both  components per pot realized the maximum vegetative  and flowering growth and oil  yield.  However, increasing salinity level of irrigated water led to  increase oil percentage.