Nine strains of Pseudomonas fluorescens and P. putida : Effects on growth indices, seed and yield production of Carthamus tinctorius L

To study the effects of growth promoting bacteria, Pseudomonas spp. on the yield production and some of the vegetative growth traits of the medicinal plant, Carthamus tinctorius variety IL111, an experiment was carried out on the Research Farm of
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  Res. on Crops 19 (4)   : 622-632 (2018) With three figures Printed in India  10 Nine strains of Pseudomonas fluorescens and P. putida : Effectson growth indices, seed and yield production of Carthamus tinctorius L. YOUSEF NASERZADEH 1, *, DAVOUD KARTOOLINEJAD 2 , NILOUFAR MAHMOUDI 1 ,MEISAM ZARGAR 1 , ELENA PAKINA 1 , MOHAMAD HEYDARI 3 ,    TAMARAASTARKHANOVA 1   AND  NYASHA JOHN KAVHIZA 1 1 Department of AgroBiotechnology Agrarian Technological Institute, RUDN University, Moscow, Russia *(e-mail :  (Received : October 12, 2018/Accepted : November 05, 2018) ABSTRACT  To study the effects of growth promoting bacteria, Pseudomonas spp. on the yieldproduction and some of the vegetative growth traits of the medicinal plant,  Carthamus tinctorius variety IL111, an experiment was carried out on the Research Farm of IlamUniversity. The experiment was laid out in a completely randomized block design (CRBD)with three replications. The treatments included the inoculation of seeds with strains 36,99, 169 and 187 of Pseudomonas fluorescens   as well as strains 168, 177, 159, 11 and 41of Pseudomonas putida   bacteria along with the non-inoculated treatment (control). In every experimental plot with 18 m² area, the inoculated seeds with bacteria were planted in sixwith an intra-row spacing of 15 cm. The results indicated that from 16 measured traitsrespecting the vegetative and yield production traits in the safflower plant, seven traitswere affected by the treatment of bacteria which included stem diameter, number of primary branches in plants, index of leaf area, plant dry biomass, crop growth rate, seed oilpercentage and the weight of 1000 seeds. These traits were superior in the plants inoculatedwith PGPR bacteria. The efficiency of P. putida   bacteria was superior to P. fluorescens   andthe control. Totally the strains 177 and 11 of P. putida   had the best vegetative and yieldproduction in terms of the mentioned traits in safflower plant. The application of biologicalfertilizers (growth promoting bacteria) demonstrated ability to improve the growthcharacteristics of this plant. Key words : Bacteria inoculation, leaf area index, Pseudomonas fluorescens  , safflower,seed oil 2 Assistant Professor of Forestry, Semnan University, Semnan, Iran. 3 M. Sc. in Analytical Chemistry, Islamic Azad University, Arak Branch, Arak, Iran. INTRODUCTION Safflower ( Carthamus tinctorius   L.)belongs to the family Asteraceae. The genus Carthamus   is comprised of 16 species and isan ancient crop (for over 4000 years) withmultipurpose uses through Asia from China tothe Mediterranean regions and along the NileValley up to Ethiopia (Bergman and Kandel2013; Hussain et al  ., 2016; Singh and Nimbkar,2016). It is grown for its medicinal properties,orange-red dye that is obtained from its petals,feed value and especially for its high quality oilwhich is rich in poly and monounsaturatedfatty acids (Dwivedi et al  ., 2005; Taleshi et al  .,2012). It has considerable resistance to bioticand abiotic stresses and can tolerate variousclimatic conditions. For the given reasons, it iscommercially cultivated in different countriessuch as India, Mexico, Canada, United States,Iran, Turkey, Uzbekistan, Kazakhstan,Ethiopia, Spain, Australia, Pakistan, Russiaand China (Singh and Nimbkar, 2016). This plant can produce proper oil whichunder optimal condition, depending on itsgenotype, can reach up to 45% (Atef, 2000).Adaptation of this plant to diverse weatherconditions of these regions is one of the  advantages of safflower plant which makes itsuitable to be cultivated in these countries andhas the capability of producing seed oil in theseareas. The environmental problems caused by excessive application of chemical fertilizers,energy, expenses of their production andconsumption, and the negative effects they have on biological cycles and self-sustainability of ecosystems are principal reasons for theapplication of the biological fertilizers (Yousefi et al  ., 2017a). Bio-fertilizers are types of fertilematerials which include one or some kinds of useful terricolous microbe species or metabolicproductions of them that are utilized for thepreparation of plant nutrients by creating asuitable symbiosis (Yousefi et al  ., 2017b;Bahmani et al  ., 2018). It is more than a century since the bacteria which enhance the growthand productivity of plants were known and theinoculum of particular species of such microbesare commonly utilized in some countries(Lugtenberg et al  ., 2001). The growth promotingbacteria are the major group of bacteria whichexist in the plant rhizosphere and due tovarious reasons cause growth increase andpromotion in plants (Yousefi et al  ., 2017a,b;Bahmani et al  ., 2018). Application of suitablebacteria in order to optimize growth of variousplant species and reduce contaminationscaused by utilization of chemical fertilizers andinsecticides, has been common practice indifferent regions in the world (Nosheen andBano, 2014). Pseudomonas fluorescens   speciesis one of the most important combination of bacterial populations in the rhizosphere(Benizri et al  ., 2001). There are various typesof non-symbiotic but useful bacteria, whichhave been introduced as the exterior root zonerhizobacteria or bacteria due to the advantageof physiological characteristics in the exteriorroot zone compared to the interior of the rootzone. These bacteria have been noticed becauseof their effects on the promotion of plant growthas well as their ability to control crop diseases.Among the various types in this group, P. fluorescens   has significant importance andbiocontrol. Pseudomonas   have been noted dueto their effects on the fertility of soil over 30 years in agricultural section. The genes of Pseudomonas spp.bacteria, belong to Pseudomonadaceae family,and the distinction of P. fluorescence   from otherPseudomonads   is the production of pigments,which against the short wavelength ultravioletlight in particular under iron deficiency condition, appears a fluorescent feature. Thesepigments with fluorescence feature andsolubility in water are called siderophores andin terms of Pseudomonas specifically calledPyoverdine or Pseudobactin (Srivastava et al  .,2012). The P. fluorescens   bacteria haveextensive distribution and excessively exist inwater, soil and rhizosphere of plantsparticularly. Various studies have been carriedout regarding this bacteria. Some of the isolatesresulted in growth increase and plant health,and consequently reduce the application of chemical materials in the agriculturaloperations. Also P. fluorescens   is one of thecommonplace bacterial species which is appliedto control plants’ phyllosphere diseases.Likewise some of the isolates of this bacteriaresult in the biological degradation of naturalcompositions or toxic compounds produced by humans and live in epiphyte form in many plants (Weller, 2007; Srivastava et al  ., 2012). P. fluorescens   strains have successfully been applied for the inoculation of seeds, potatoroot ( Solanum tuberosum   L.), sugar beet root( Beta vulgaris   L.), wheat ( Triticum aestivum   L.)and in some other crops (Weller and Cook,1986; Dowling and Ogara, 1994).Considering the expansion of theapplication of organic compounds in agricultureas well as the increase in production demandfor oil plants, the objective of this research isthe verification of the influence of P. fluorescens  and P. putida   bacteria strains on the vegetativegrowth and yield production traits of safflowerplant. MATERIALS AND METHODS In order to the study of the effects of growth promoting bacteria ( P. fluorescens  )application on the yield production andvegetative growth traits of safflower plant,variety IL111, an experiment was carried outon the research farm of the agriculture faculty of Ilam university in fall 2016. The researchfarm is located latitude of 31°58 ′ N, longitudeof 45°24 ′ E, and altitude of 1427 m above sealevel. The experimental region has semi-dry weather with warm and dry summers. This research was carried out in acompletely randomized block design (CRBD) Effect of Pseudomonas strains on safflower  623  with 10 treatments and three replications. Thetreatments included the inoculation of safflowerseeds with four strains of P. fluorescens   bacteria(strains 36, 99, 169 and 187) and five strainsof Pseudomonas putida   (strains 11, 41, 159,168 and 177) along with the control (withoutinoculation). These strains of bacteria providedfrom the central soil biology institution of Iran.In order to execute the inoculation of seeds,half an hour prior to sowing in the field, theseeds were put in a shady place far fromsunlight and wind. The seeds of each treatmentwere wetted by spraying some water, thenbecame sticky by the gum Arabic solution andthe inoculation substance was added into theseeds which included the powder saucecontaining the bacteria strains (the nutrientagar for the nourishment of bacteria was alsoput in). Afterwards, it was shaken for 30 sec tomix all seeds with the inoculum homogenously and immediately after inoculation all seedswere planted in the plots. The area of each experimental plot was18 m² (5 × 3.6 m) in which the plants werecultivated in six rows with an inter-row spacingof 60 cm. The intra-row spacing was 15 cm.Between two neighbouring treatments, one rowwithout planting was considered as a buffer. The distance between blocks was 4 m. Thesowing of seeds was done manually in the rowsat a depth of 3 cm. The thinning operation was carried outwhen the plants had 5 to 6 leaves and theheight was 15 up to 20 cm. The first irrigationwas done on 5 November 2016 and this datewas considered as the planting date. In the nextirrigation cycles, the watering interval wasseven days. Weeding was performed manually and simultaneously in all treatments.Harvesting of the safflower was carried outmanually as soon as the yellowing sign of leavesand fruits was observed on 23 July 2017.For the determination of growthparameters and indicators, 15 plants werechosen randomly from each plot, but from twocentral rows of each plot so as to eliminate theborder effect (half a meter from the beginningand half a meter from the end of the plot). Thenthe traits such as the number of the main andsecondary branches in each plant, plant height,stem diameter, leaf area, number of fruits perplant, number of seeds per fruit and the weightof 1000 seeds were determined. The seed yieldand harvest indicator per unit area werecalculated with the surface area of 2 m² fromtwo central rows of the first half part of theplot in the exception of half a meter bordereffect. For the measurement of the weight andpercentage of seed husk, 20 seeds were weighedand afterwards transferred into the germinationpetri dishes and then kept at 25°C withsufficient quantity of humidity in the cultivationchamber for 48 h. After that time due to thegermination of the seeds, the seed husk wasseparated easily. The husks of each treatmentwere transferred into paper pockets andmaintained at the temperature of 75?C forabout 48 h in the oven to dry out. Afterwardsthey were weighed again and by the subtractionof seed coat weight from total seeds weight,the weight of kernel was calculated.Seed coat(%)=Seed coat dry weight/ Total seed weight × 100 ...(Eq. 1)Safflower seed oil percentage wasdetermined by Soxhlet method, 1 g of grindedseed extracted for oil by utilization of Hexaneorganic solvent for 4 h and eventually by equation 2 the oil percentage of safflower seedswas calculated. Oil per cent=(W 3  –W 2 /W 1 ) × 100 ...(Eq. 2)Where, W 1 : Sample weight, W 2 : The initialweight of the glass and W 3 : Thesecondary weight of glass The percentage of proteins wascalculated by Kjeldahl method with applicationof 1 g samples taken from each plot and grindedin the laboratory and eventually by equation 3the quantity of nitrogen or raw oil percentageof the sample was calculated.Percentage of raw protein in the dry matter= (raw protein per cent/100)– Moisturepercentage) × 100 ...(Eq. 3)Ultimately the protein productivity wascalculated by multiplying the seed proteinpercentage for the seed yield of each plot. TheSAS software was used for the analysis of data(SAS Institute, 2012) and the comparison of means was carried out by Tukey’s HSD test at5% level of probability.624 Naserzadeh, Kartoolinejad, Mahmoudi, Zargar, Pakina, Heydari, Astarkhanova and Kavhiza   RESULTS AND DISCUSSIONVegetative Characteristics of Safflower PlantNumber of main and secondarybranches in plant :  Analysis of variance(ANOVA) indicated that the plants undervarious seed inoculation treatments hadsignificantly higher numbers of main branchesper plant at 5% level of probability. The groupcomparison indicated that the treated plantswith Pseudomonas putida   bacteria (strains 177and 11 in particular) in comparison to othershad better productivity (Tables 1 and 2).In terms of the number of the secondary branches, P. putida   bacteria versus the controlcompared to P. fluorescens   bacteria versus thecontrol with 28% precedence had the maximumvalue (Table 3).Cheng et al  . (2007) indicated thatbacteria genes putida with deaminase-ACCenzyme production ability resulted in theincrease in the number of safflower branches. Stem diameter and plant height :  Theeffect of inoculation with bacteria on the meanof safflower stem diameter was significant(P>0.05) (Table 1). Plants treated with P. putida  bacteria had the maximum stem diameter andthe control (without inoculation) had theminimum. The plants treated with P. putida  bacteria in comparison with the ones treatedwith P. fluorescens   bacteria had 5% precedence(Table 3). According to the studies of researchers, P. putida   bacteria with moreactivity facilitated the plant growth by varyingthe hormonal level and producing auxinhormone in some parts of the plant, resultingin cell elongation, cell division, differentiationof roots and consequently caused the increase Table 1.  Analysis of variance†, safflower vegetative traits between the bacteria inoculation treatmentsVariations/resourcesd. f.No. of mainNo. of secondaryPlantStembranches in plantsbranches in plantsheightdiameterBlocks20.50 ns 8.28 ns 55.47 ns 0.41 ns  Treatments91.00*2.57 ns 21.37 ns 0.90*Error180.965.7242.960.38C. V. (%)-17.2822.167.799.60†Values in the variance analysis table are based on ms. *Significant difference at P=0.05 level. NS : Not Significant. Table 2.  Mean comparison of the vegetative growth traits of safflower between the bacterial inoculation treatments TreatmentNo. of mainNo. of secondaryPlantStembranches in plantsbranches in plantsheightdiameterControl (without bacterial inoculation)4.7b5.3a82.9a5.7abStrain 36  P. fluorescence  5.6ab8.0a85.4a6.3abStrain 99 P. fluorescence  5.6ab7.2a85.4a6.6abStrain 169 P. fluorescence  5.2ab5.4a77.7a5.8abStrain 187 P. fluorescence  5.3ab6.1a85.9a6.6abStrain 11 P. putida  5.8ab7.1a84.8a7.1aStrain 41 P. putida  6.4ab7.5a83.5a6.5abStrain 159 P. putida  5.5ab6.5a82.9a5.7abStrain 168 P. putida  5.8ab8.5a84.5a6.6abStrain 177 P. putida  6.7a7.3a87.7a7.2a The different letters in each column indicate the significant differences at P=0.05 probability level. Table 3.  The results of the variance analysis† of safflower vegetative growth traits for the comparison of the mean in thetreatment groups TreatmentNo. of mainNo. of secondaryPlantStembranches in plantsbranches in plantsheightdiameterAll bacterial strains vs. control3.65 ns 8.85 ns 4.80 ns 1.39 ns P. putida   vs. P. fluorescens  2.46 ns 3.08 ns 6.93 ns 0.95 ns P. putida vs. control5.13*11.16 ns 7.98 ns 1.96* P. fluorescens   vs. control1.63 ns 4.93 ns 1.41 ns 0.62 ns†  The values in the variance analysis table are based on ms. *Significant at P=0.05 level. NS : Not Significant. Effect of Pseudomonas strains on safflower  625  in stem diameter, stem length, ethylenebiosynthesis and change in the expression of particular genes (Rolfe et al  ., 1997; Jalili et al  .,2011). The various treatments did not havesignificant effect on the height of safflower plantin this research (Tables 1 and 2). While Leoni et al  . (2002) attributed the positive effect of P. putida   bacteria application in the heightincrease of the corn plant to production of growth hormones such as auxin as well assiderophores by these bacteria. Mirzashahi(2012) also reported the increase in the heightof wheat inoculated with growth promotingbacteria. Leaf area index (LAI) :  As it isdemonstrated in Fig. 1, the trend of changesin leaf area index was generally similar, and itwas an indicator for the increasing trend butsomehow slow in all treatments, 180 days afterplanting. Nearly 192 days after planting, theincrease in LAI was due to the increase in thenumber of leaves and leaf area. In this stage,the plants treated with P. putida   bacteriastrains 177 and 168 had the maximum andthe control plants (without inoculation) theminimum LAI (Fig. 1). The increasing trend inthe leaf area index continued until 216 daysafter planting, so that in this stage the plantstreated with P. putida   bacteria strains 177 and11 had the maximum and the plants withoutinoculation (59% lower) the minimum LAI.Afterwards the decreasing trend started in theleaf area index, so that after 228 days thetreated plants by strains 177 and 11, againhad the maximum and the control had theminimum leaf area index. According to thehypothesis of Fracis et al  . (2000), the maximumLAI in the inoculated plants, was apparently because of the more activity of these bacteriaand nitrogen fixation, and consequently hadmore leaf durability.According to the statements of Zaidi(2003), the reason for growth increase becauseof the presence of P. fluorescens   was declaredthe changes in the concentration of growthregulator compounds such as cytokinin,gibberellin and ethylene. Plant dry mass :  Early in the growingseason, plant dry weight was very low until 180days after planting due to low temperature andphotosynthetic levels in all treatments. At thesame time, the treated plants with P. putida  bacteria strain 177 produced the maximumamount of dry matter compared to othertreatments, hence began the linear growthfaster (Fig. 2). The minimum production of dry biomass throughout the growing period wasobserved in the control treatment and P. putida  strain 159. After 216 days from the plantingdate, the plants treated with P. putida   strain11 had the maximum accumulation of dry matter. So the trend of plant dry biomassaccumulation was linear and ascending untilday 216 after planting. After 228 days from theplanting date, the plants treated with P. putida   ‐  1   2   3   4   5   6180 192 204 216 228       L     e     a       f      A     r     e     a      I     n       d     e     x Days   after   planting   169   187   11   177   41   99   36   168   159 Control Fig. 1. Safflower plant leaf area index under the effect of various treatments of bacterialinoculation. 626 Naserzadeh, Kartoolinejad, Mahmoudi, Zargar, Pakina, Heydari, Astarkhanova and Kavhiza 
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