journa l h om epage: www.elsev ier .com/ locate /apsoi l
ea growth and symbiotic activity response to Nod factorslipo-chitooligosaccharides) and soil compaction
nna Siczeka,∗, Jerzy Lipieca, Jerzy Wielbob, Paweł Szarlipa, Dominika Kidajb
Institute of Agrophysics, Polish Academy of Sciences, P.O. Box 201, 20-290 Lublin, PolandDepartment of Genetics and Microbiology, M. Curie-Skłodowska University, Akademicka 19 Street, 20-033 Lublin, Poland
r t i c l e i n f o
rticle history:eceived 8 January 2013eceived in revised form 28 June 2013ccepted 29 June 2013
Growth and symbiotic activity of legumes are reduced by high soil compaction and mediated by Nodfactors (LCO, lipo-chitooligosaccharides) application. Our objective was to assess the combined effectsof soil compaction and Nod factors application on growth and symbiotic activity of pea. The experimentwas two factorial and included soil compaction (1.30 g cm−3 – not compacted (control) and 1.55 g cm−3
– compacted soil), and Nod factors concentration (control without addition of Nod factors and use of260 nM Nod solution) for each soil compaction. The soil (Haplic Luvisol) was packed into pots, pea (Pisumsativum L.) seeds were soaked with Nod factors solution or water and then plants were grown for 46 days.This study has shown that soil compaction and treatments of pea seeds with Nod factors influenced peagrowth and symbiotic activity. Soil compaction significantly reduced pea growth parameters, namelyplant height, dry mass, leaf area, root mass and root length and symbiotic parameters, namely mass of
lant nitrogen content nodules, dry mass of an individual nodule, nitrogenase activity and total nitrogen content in plant incomparison to the non-compacted treatment. Treatment of seeds with Nod factors generally improvednearly all of the above parameters. Nitrogenase activity per pot and total plant nitrogen content weresignificantly reduced by soil compaction and increased by application of Nod factors in plants grown innot compacted soil. Our results demonstrate that increased symbiotic activity resulting from Nod factorsaddition may mitigate adverse effect of soil compaction on plant growth.
Excessive soil compaction mostly results from intensive crop-ing and no-tillage practices, heavy vehicle and implement traffic.
t harmfully influences many soil characteristics (Hamza andnderson, 2005; Horn et al., 2003) especially shifting pore sizeistribution to smaller pores and, consequently, activity and diver-ity of soil microorganisms (Nosalewicz and Nosalewicz, 2011;engthamkeerati et al., 2011; Siczek and Frac, 2012) and plantrowth (Gregorich et al., 2011). Soil compaction may alter themount of nitrogen that is supplied to agricultural system by asso-iation of nitrogen fixing bacteria with legume. Soil compactionnfavorably affects grain and protein yield of soybean (Botta et al.,010). The effect of soil compaction on nodulation and nodulectivity can be mediated by soil moisture status (Lindemann
t al., 1982) and mulch applications (Siczek and Lipiec, 2011).he results of Buttery et al. (1998) showed that increasingoil moisture alleviated the effect of compaction on nodulation
of soybean and common bean in sandy loam. Voorhees et al.(1976) reported that soil compaction altered the distribution ofthe nodules in the vertical and horizontal plane. The study ofSiczek and Lipiec (2011) showed that the greatest total nod-ule number and weight occurred in moderately compacted soilwhereas the contribution of large nodules and the dry weightof individual nodules were the greatest in the most compactedsoil.
Legumes are important grain and forage crops in both tem-perate and tropical climates. Due to symbiotic association withgram-negative soil bacteria called rhizobia, they depend slightlyon soil nitrogen status. Abi-Ghanem et al. (2011) reported that theproportion of plant N supplied by fixation varied with pea vari-ety and ranged from 87% to 91% in a pot experiment. The fieldstudy of Hauggaard-Nielsen et al. (2010) showed that the percentof total N derived from the atmosphere at flowering in pea rangedfrom 65% to 92% with quantitative N2-fixation estimates from 93to 202 kg N ha−1. At maturity these values decreased respectively
to 26–81% and 48–167 kg N ha−1. Considering the dry spring anda major rainfall event around flowering, it is likely that a high netmineralization of soil N took place causing the increased uptake ofsoil N and reduced fixation.
182 A. Siczek et al. / Applied Soil Ecology 72 (2013) 181– 186
Table 1Characteristics of the soil plough layer.
Clay Silt Sand (g kg−1) C org Total N* P** K (mg kg−1) Mg pH H2O
70 290 640 14.1 0.75 90 153 23 5.9
* As indicated by the Kjeldahl method.**
m2atiCmptrtetldaDte(gfaAbstolAtaie
babbXr
cfc
2
2
f(hc
Plant available inorganic P.
Symbiotic association between legumes and rhizobia involvesolecular signal exchange between both partners (Dardanelli et al.,
012; Hirsch et al., 2001). Plant roots excrete many compounds thatre chemoattractans for the rhizobia. Among them flavonoids arehe strongest inducers of nod genes. The main role of flavonoidsn the initiation of symbiosis is interaction with the NodD protein.omplex flavonoids-NodD protein induces the production of keyolecules that allow the plant host recognition of the bacterial
artner, the Nod factors (LCO, lipo-chitooligosaccharides). Nod fac-ors consisting of a skeleton of three to five N-acetyl d-glucosamineesidues linked to a lipid moiety on the nonreducing end. Nod fac-ors induce many early nodulation events that occur at the plantpidermis, cortex and pericycle. At submicromolar concentrationshese molecules are responsible for membrane potential depo-arization, formation of infection threads, root hair deformation,ivision of root cortex cells and formation of nodule primordia,nd induction of early nodulin gene expression (for review see’Haeze and Holsters, 2002). The study of Maj et al. (2009) indicated
hat pretreatment of red clover seeds with Nod factors significantlynhanced plant nodulation and growth. More recently, Kidaj et al.2012) confirmed a beneficial effect of Nod factors on pea and vetchermination, nodulation and growth. The foliar application of Nodactor to soybean significantly (P < 0.05) increased leaf area, shootnd dry mass compared with control plants (Khan et al., 2008).lmaraz et al. (2007) also demonstrated positive response of soy-ean to some concentrations of Nod factors. In the above quotedtudy photosynthesis was increased up to 13% over the control, andhis was connected with increases in plant dry weight. It has beenbserved that Nod factors may induce seed germination in non-egume plants such as barley (Miransari and Smith, 2009) corn andrabidopsis thaliana (Prithiviraj et al., 2003). It has been recognizedhat addition of Nod factors affected early events of the symbiosisnd growth of legume crops in stressed growth conditions includ-ng low rhizosphere temperature, low pH and water stress (Attit al., 2005; Duzan et al., 2004, 2006; McKay and Djordjevic, 1993).
Recent evidence suggests roles for rhizobial Nod factors that goeyond the nodulation process. Duzan et al. (2005) showed thatpplication of the Nod factor Nod Bj-V (C18:1, MeFuc) induced soy-ean resistance to powdery mildew caused by Microsphaera diffusay fungal growth and development hampering. On the other handie et al. (1998) reported that application of Nod factors to legumeoots promoted arbuscular mycorrhizal fungi (AMF) colonization.
There is currently little evidence on combined effects of soilompaction and Nod factors on the plant growth parameters, there-ore our objective was to investigate whether Nod factors treatmentan mediate soil compaction under the same growth conditions.
. Materials and methods
.1. Soil and growth conditions
The soil, a Haplic Luvisol developed from loess, was collected
rom the plough layer (0–20 cm) in an arable field in Lublin, Poland51◦13′ N, 22◦37′ E). Soil was collected in the autumn after wheatarvest and before tillage operations, and at a soil water contentorresponding to field capacity. The soil at the sampling site was
under long-term (30 years) conventional tillage, with main tillageoperations including pre-plough (10 cm depth) and harrowing, andmouldboard ploughing (20 cm depth). Characteristics of the soilplough layer are presented in Table 1.
After sampling, the soil was sieved through a 0.4 cm sieve. Thesoil was packed into PVC pots 15 cm in diameter and 40 cm high totwo bulk densities: 1.30 g cm−3 – not compacted soil (control), withbulk density optimal for plant growth in this soil, and 1.55 g cm−3 –compacted soil with bulk density that is considered as the limitingfor plant growth and function. A known weight of soil was com-pacted into successive 2 cm layers with a piston that was pressedby a hydraulic press to get bulk density 1.30 g cm−3 and 1.55 g cm−3,respectively. Before packing into pots, soil at layers of 0–20 cm wasuniformly mixed with fertilizers in amounts corresponding to P,K and Mg of 40, 50 and 35 kg ha−1, respectively. The number ofRhizobium leguminosarum bv. viciae in the plough layer of the sam-pled soil amounted to 1.8 × 103 per g of dry soil as determined usingthe most probable number technique modified by Martyniuk et al.(2000) and was assessed as high (Martyniuk et al., 2005). Due tothis fact, in this study pea seeds were not inoculated with R. legu-minosarum bv. viciae.
The experiment was designed as a 2 × 2 factorial. The firstfactor was soil compaction (1.30 g cm−3 – not compacted (con-trol) and 1.55 g cm−3 – compacted soil) and second factor wasNod factors concentration (control without addition of Nod factorsand use of 260 nM Nod solution). Pea (Pisum sativum L. cv. Tar-chalska) seeds were soaked for 30 min with Nod factors solution260 nM or water. Then six seeds per pot were placed at 3 cm soildepth. After emergence seedlings were thinned to three per pot.Each of the four treatments: NC (not compacted, without Nod fac-tors), NC + Nod (not compacted, with Nod factors), C (compacted,without Nod factors) and C + Nod (compacted, with Nod factors)were replicated four times. The pots were arranged randomly ina controlled growth-chamber environment. Day-time (16 h) andnight-time (8 h) temperatures were 22 ◦C and 17 ◦C, respectively,relative humidity (RH) was 65 ± 5% and photosynthetically activeradiation (PAR) amounted to 320 �mol m−2 s−1. During pea growthsoil moisture was kept on a level optimal for plant growth, corre-sponding to soil water potential −31 kPa (pF 2.5), by measuring theevapo-transpired water three times a week (calculated by weigh-ing the pots) and replenishing the lost water. The experiment wasstarted on 9th September 2011. Plants were grown for 46 days upto the flowering stage.
2.2. Preparation of exudates from sprouted seeds
Pea seeds were surface sterilized by immersion in 0.1% HgCl2for 3 min, rinsed with sterile distilled water, then treated with70% ethanol for 3 min, followed by a sterile water wash. Then, theseeds were shaken in sterile water, in darkness, for four days at28 ◦C. Sprouted seeds were separated from water suspension afterdecantation, and plant tissue debris were removed from the super-
natant by centrifugation (4000 rpm, 10 min). Then ethyl acetatewas added to the supernatant (1:10, v/v), and flavonoids wereextracted with ethyl acetate from the aqueous phase in a separatingdropping funnel. After that ethyl acetate was evaporated, and the
oil Ecology 72 (2013) 181– 186 183
psicu
2
gO(stb4tabaTasoNflbbtrhwtartwtv5mw(csoi
2
s(gnataptibGTsa
0%
25%
50%
75%
100%
NC NC+Nod C C+Nod
Freq
uenc
y
ab
aa
aa
aa
aba
aa
b>10 mm2 5-10 mm2 <5 mm2
Fig. 1. Effects of soil compaction and Nod factors application on nodule area distri-
A. Siczek et al. / Applied S
ellet containing flavonoids was resolubilized in 95% ethanol andtored at 4 ◦C. The amount of the flavonoids was determined by dry-ng and weighing the ethanol extract. The approximate flavonoidoncentration in seed extracts was calculated relative to the molec-lar weight of authentic flavone (Maj et al., 2010).
.3. Nod factors production and isolation
5 ml R. leguminosarum bv. viciae strain GR09 cultures wererown overnight in liquid TY broth (Sambrook et al., 1989) to anD550 of 0.6 and was then used to inoculate five 200 ml aliquots
in 400 ml flasks) of liquid TY medium (1:200, v/v). The synthe-is of rhizobial Nod factors was induced by adding peas seeds tohe flavonoids at a final concentration of 10 �M, and then rhizo-ial cultures were incubated with shaking (170 rpm) at 28 ◦C for8 h. To isolate Nod factors, one liter of the flavonoid-induced cul-ure was centrifuged (5000 rpm, 10 min) to remove bacterial cells,nd the supernatant was extracted twice with 0.2 volume of n-utanol (Prithiviraj et al., 2003). The organic fraction was separatednd dried in a rotary evaporator (Rotavapor-R, Buchi, Switzerland).he amount of Nod factors was determined by conversion of themino sugars to methyl glycosides and gas chromatography/masspectrometry (GC/MS) analysis (Maj et al., 2009). 250 �g of driedrganic fraction was mixed with internal standard, i.e., 30 �g of-acetylgalactosamine (GalNAc) and hydrolyzed in 1 ml of 2 M tri-uoroacetic acid (TFA) at 120 ◦C for 2 h. The TFA was eliminatedy washing the samples two times with Millipore water, followedy drying under nitrogen. Free fatty acids were removed fromhe samples with 0.5 ml 10% (v/v) ether in hexane. This step wasepeated three times. Samples were reduced by solid sodium boro-ydride (NaBH4) at 0 ◦C, in darkness, for 12 h. The excess NaBH4as removed by sequential distillation with the following solu-
ions: 200 �l 10% (v/v) acetic acid in water, 200 �l 10% (v/v) aceticcid in methanol, and 200 �l 1 M HCl in methanol. Samples wereedistilled two times with methanol and dried under nitrogen. Inhe next acetylating step, 25 �l acetic anhydride and 50 �l pyridineere added to the dry sample and incubated at 85 ◦C for 30 min and
hen dried under nitrogen. The samples were dissolved in a smallolume of chloroform and applied to GC/MS (Hewlett-Packard HP890A) equipped with an HP.5MS capillary column coupled to aass selective detector MSD HP 5971. The temperature programas as follows: initially 150 ◦C for 5 min, then raised to 310 ◦C
5 ◦C/min), and the final temperature 310 ◦C for 10 min. Nod factorsoncentration was approximated based on the assumption that aingle molecule of a Nod factor contains on average four residuesf N-acetylglucosamine (GlcNAc). The calculated content of GlcNAcn the Nod factors preparation was 260 nM.
.4. Plant measurements
At the flowering stage plants were harvested and analyzed forhoot height, dry mass (after drying at 65 ◦C for 48 h) and leaf areaDelta-T Image Analysis System). Roots with attached nodules wereently separated from the soil and washed on a 0.4 mm sieve. Thenitrogenase activity measurements were conducted by means ofn acetylene reduction assay (C2H2). This technique was valuableo assess differences in the N fixation rate among soil compactionnd mulching treatments (Siczek and Lipiec, 2011). Roots from eachot were placed in bottles (one pot per one bottle) and 10% (v/v) ofhe gas from the bottles was replaced with acetylene. After 30 minncubation at room temperature 1 ml of gas was sampled from theottles and analyzed for ethylene concentration using a Shimadzu
C14-B gas chromatograph with flame ionization detector (FID).he 274.3 cm long stainless steel column was packed with carbo-phere 80/100 mesh. The temperatures of the injector, column ovennd detector were 150 ◦C, 230 ◦C and 230 ◦C, respectively. Helium
bution. Bars represent standard errors (n = 4). Different letters within each nodulearea indicate significant differences (P < 0.05). NC: not compacted soil, C: compactedsoil, Nod: Nod factors.
was used as the carrier gas. The amount of ethylene (�mol h−1)as measured per pot was converted to 1 g of nodule dry weight(specific nitrogenase activity).
The number, area and dry mass of nodules were determined foreach pot. Nodules and roots were scanned separately with a reso-lution of 600 dpi (dots per inch). The area distribution of noduleswas calculated using ImageJ program (National Institute of Health,USA) whereas root length and average diameter with the Win-RHIZO 2007 (Regent Instruments Inc.) program. Nodule and rootdry mass was obtained by following the same drying procedure asused for the determination of the shoot mass. Specific root length(SRL) was calculated by dividing root dry mass by length.
Total plant mass was calculated by summarizing dry mass ofshoots, roots and nodules and was expressed in g per pot. Nitrogencontent was determined separately in pea leaves, stems, roots andnodules by means of the Kjeldahl method in three replications.
2.5. Statistical analysis
Statistical analysis was performed using STATISTICA 8.0 (Stat-Soft, Inc.). Data were analyzed by two ways ANOVA. When ANOVAresulted in a significant F test, then mean separation analyses wereperformed (post hoc test Tukey HSD).
3. Results
3.1. Compaction effects on plant growth and nodulationcharacteristics
Soil compaction significantly (P < 0.05) reduced mean values ofanalyzed shoot parameters (height, dry mass and leaf area by 9%,36% and 42%, respectively) in comparison with NC (Table 2). Pearoot mass and length were also significantly reduced due to com-paction, however, root average diameter increased significantly(P < 0.05) by 21% due to compaction as shown by the values ofspecific root length (SRL) that decreased by 39%.
The results demonstrated a harmful effect of compaction on themean values of nodule mass per pot and on the mass of an individualnodule. Nodule mass per unit root mass was significantly reduced(41%) in C in relation to NC. Mean values of total plant dry massdecreased due to compaction by 36%. Compaction affected signifi-cantly nearly all shoot and root parameters (Table 3), and generallythere were no combined effects between compaction and Nod.
The data in Fig. 1 show that soil compaction affected the dis-tribution of the nodule area. In general, compaction resulted in agreater share of nodules with an area of <5 mm2 and lower shareof nodules of >10 mm2, in comparison with NC. The difference in
184 A. Siczek et al. / Applied Soil Ecology 72 (2013) 181– 186
Table 2Mean values of pea shoots, roots and nodules parameters. Different letters for means within each source of variability (compaction and Nod factors) indicate significantdifferences (P < 0.05). SRL: specific root length.
Plant parameters Compaction* Nod factors**
Not compacted Compacted Without Nod factors With Nod factors
Shoot (plant−1)Height (cm) 76.7 a 69.7 b 71.6 b 74.8 aMass (g) 4.85 a 3.10 b 3.70 b 4.25 aLeaf area (cm2) 267.1 a 153.7 b 199.3 b 221.5 a
Roots (three plants−1)Mass (g) 1.36 a 0.89 b 1.04 b 1.21 aLength (m) 173.8 a 67.9 b 112.6 b 129.1 aSRL (m g−1) 128.6 a 77.8 b 103.6 a 102.8 aDiameter (mm) 0.330 b 0.417 a 0.376 a 0.371 a
Nodules (three plants−1)Number 122.1 a 94.8 a 102.0 a 114.9 aMass (mg) 223.3 a 86.1 b 148.0 a 161.3 aMass (mg nodule−1) 2.34 a 0.98 b 1.65 a 1.66 aNodule mass (mg)/root mass (g) 165 a 98 b 135 a 128 aTotal plant mass (g three plants−1) 16.01 a 10.23 b 12.23 b 14.02 a
nw
crniscw
3c
b
TRf
N
0
10
20
30
40
50
thre
e pl
ants
-1b
bcc
ase
ac�
vity
(C2H
4μm
ol h
-1)
200
ght
* Compaction include treatments with and without Nod factors.** Nod factors include treatments with and without compaction.
odules with an area of >10 mm2 between NC + Nod and C + Nodas significant (P < 0.05).
As indicated in Fig. 2, nitrogenase activity per pot was signifi-antly (P < 0.05) lower (by 48.3%) in C than NC. However, an oppositeesponse to soil compaction was recorded with respect to specificitrogenase activity (per 1 g nodule dry weight), it increased signif-
cantly with compaction. Both nitrogenase activity indicators wereignificantly affected by compaction (Table 3). Total plant nitrogenontent significantly (P < 0.05) decreased under C in comparisonith NC (Fig. 3).
.2. Nod factors effects on plant growth and nodulation
haracteristics
Nod factors significantly improved height, dry mass and leaf areay 4%, 13% and 10%, respectively (Table 2). Pea root mass and length
able 3esults of two-way ANOVA of plant characteristics against soil compaction (C), Nod
S – not significant. SRL: specific root length.* P < 0.05.
** P < 0.01.*** P < 0.001.
Nitr
ogen
a
0
50
100
150
NC NC+Nod C C+Nod
g -1
nod
ule d
ry w
ei
c
abb
Fig. 2. Effects of soil compaction and Nod factors application on nitrogenase activity.Bars represent standard errors (n = 4). Different letters indicate significant differ-ences (P < 0.05). NC: not compacted soil, C: compacted soil, Nod: Nod factors.
0
0.2
0.4
0.6
NC NC+Nod C C+Nod
Tota
l pla
nt n
itrog
en c
onte
nt
(g N
thre
e pl
ants
-1) b
cc
a
Fig. 3. Total plant nitrogen content as related to soil compaction and Nod factorsapplication. Bars represent standard errors (n = 4). Different letters indicate signif-icant differences (P < 0.05). NC: not compacted soil, C: compacted soil, Nod: Nodfactors.
A. Siczek et al. / Applied Soil Eco
y = 0.0132 x + 0.128 5R² = 0.88 1
0
0.2
0.4
0.6
0 10 20 30 40
Tota
l pla
nt n
itrog
en c
onte
nt
(g
N th
ree
plan
ts-1
)
Nitrogenase ac�vity (C2H4 μmol h-1 three plants -1)
FA
wdTaufHrt
(inissg(u
3n
ccts
4
4
ttr1dis
gwcssoTi
ig. 4. Relationship between nitrogenase activity and total plant nitrogen content.ll study treatments were included.
ere also significantly improved by Nod application. Root averageiameter remained nearly unchanged in response to Nod factors.he effects of Nod factors on nodule parameters (number and mass)nd nodule mass per unit root mass were not significant. Mean val-es of total plant dry mass increased due to Nod factors by 13%. Nodactors affected significantly most of the plant parameters (Table 3).owever, significant interaction between compaction and Nod for
oot diameter may result from significant effect of compaction onhis parameter rather than from Nod factors (insignificant effect).
Nod factors slightly affected the distribution of the nodule areaFig. 1). It increased share of large nodules with an area of >10 mm2
n NC and decreased in C. Nod factors led to an increase in nitroge-ase activity per pot under NC by 56 and under C by 33% (Fig. 2). This
ncrease was statistically significant (P < 0.05) under NC. Similarly,pecific nitrogenase activity (per 1 g nodule dry weight) increasedignificantly with Nod application both under NC and C. Both nitro-enase activity indicators were significantly affected by Nod factorsTable 3). Nod factors favorably affected total plant nitrogen contentnder NC (Fig. 3).
.3. Nod factors and compaction effects on plant growth andodulation characteristics
Soil under C with Nod factors showed significant increase in spe-ific nitrogenase activity (Fig. 2) and decrease in total plant nitrogenontent (Fig. 3) compared to NC without Nod factors. The distribu-ion of nodule area (Fig. 1) and nitrogenase activity per pot not differignificantly between these treatments (Fig. 4).
. Discussion
.1. Compaction effects on pea growth and symbiotic activity
The results from this study demonstrated a significant reduc-ion in nearly all analyzed plant parameters in compacted relativelyo not compacted soil. With respect to plant mass, leaf area andoot elongation these results confirm literature data (Buttery et al.,998; Lipiec et al., 2012). The field study of Siczek and Lipiec (2011)emonstrated significantly lower soybean seed and protein yields
n highly compacted soil in comparison with medium compactedoil.
In this study, we observed a substantial decline (by 48%) of nitro-enase activity under C compared to NC. Lower nitrogenase activityas connected with both lower nodule number and mass in C in
ontrast to NC, although this was partly compensated for by higherpecific nitrogenase activity (per 1 g nodule dry weight). Compen-
ation of specific nitrogenase activity was also observed under lowxygen concentration in the root zone (Dakora and Atkins, 1991).his decline can be due to structural alterations in nodules includ-ng: increase in volume of cortex relative to the infected central
logy 72 (2013) 181– 186 185
tissue and an alteration in the distribution of infected and unin-fected cells in the central tissue zone. Structural changes of noduleswere observed under oxygen deficiency, reduced nitrate avail-ability and water stress (Dakora and Atkins, 1991; Mrema et al.,1997). Reduced pore size and associated increased soil mechani-cal impedance appear to be the main factors responsible for thechanges in pea growth and symbiotic activity under compactedtreatment in relation to not compacted treatment. Some earlierstudies stressed that the nitrogenase activity of legume plants wasreduced under anoxic conditions (Dakora and Atkins, 1991; Guaschet al., 2001). A negative effect of high soil compaction on nitroge-nase activity was stated for soybean by Lindemann et al. (1982) andSiczek and Lipiec (2011). In the present study, soil porosity underC was lower than under NC (14.0% vs. 23.1%), but both values wereabove 10% (v/v) which is considered critical to allow root growth(Lipiec and Hatano, 2003).
4.2. Nod factor effects
The results demonstrated an improvement of pea growth due tothe treatment of seeds with Nod factors. The effect of Nod factorswas significant (P < 0.05) for nitrogenase activity, shoot height andmass, leaf area, root mass and length and total plant mass and nitro-gen content. Better plant growth under Nod factors treatments thancontrol treatments (without Nod) corresponds well with greater Ncontent in the plant biomass due to higher nitrogenase activity. Ahigh correlation (R2 = 0.881) was found between nitrogenase activ-ity and total plant N content as well as nitrogenase activity and totalplant mass (R2 = 0.796).
Our results imply generally better roots than shoots growthafter pea seed inoculation with Nod factors. More pronounced Nodfactor effects for the roots than shoots were observed earlier byother researchers (Maj et al., 2009; Souleimanov et al., 2002). Majet al. (2009) stated a more robust root growth (1.64-fold) of cloverin response to Nod addition than shoots (1.37-fold) in relation tothe control plants. Nod factors improved soybean root growth byincreasing root length, surface area and dry weight (Souleimanovet al., 2002) and stimulated lateral root formation in Medicago trun-catula (Oláh et al., 2005). The beneficial effect of Nod factors onpea growth and symbiotic activity can be a result of mitogenicand morphogenic activity leading to induction of cell cycle genesand cell division (Khan et al., 2008; Prithiviraj et al., 2003). It isknown that Nod factors promote early somatic embryogenesis innon-legume plants, even in the absence of auxin and cytokinin(Dyachok et al., 2000; Egertsdotter and von Arnod, 1998), seed ger-mination and early growth of plants (Miransari and Smith, 2009;Souleimanov et al., 2002). Enhanced growth rate was connectedwith increased ˛-amylase activity in the seeds of Nod-treated corn,which would have triggered more rapid breakdown of endosperm-stored starch reserves (Prithiviraj et al., 2003). Another possiblereason for improved plant growth induced by Nod factors mightresult from its influence on the localized changes in phytohormones(auxin and cytokinin) balance and various physiological responsesin plants (Mathesius et al., 1998). They also stated that endoge-nous flavonoids could mediate Nod factors response. Schmidt et al.(1994) observed an increase in flavonoids concentrations in the rootexudates of soybean following treatment with Nod factors.
Nod factor effects on nodule activity could be also partiallyconnected with influence on symbiotic competitiveness of the bac-terial strains. Recently, Kidaj et al. (2012) observed that Nod factorsslightly influenced the level of genetic diversity of rhizobia col-
onizing both pea and vetch nodules. They suggested that Nodfactors added to the seeds influence symbiotic compatibility ofthe microsymbiont and could affect the recruitment of indigenousrhizobia for colonization of emerging nodules.
1 oil Eco
fsss
5
np(d
(
(
(
A
f2
R
A
A
A
B
B
D
D
D
D
D
D
D
E
86 A. Siczek et al. / Applied S
To our knowledge this is the first study assessing effects of Nodactors in relation to soil compaction. These results suggest the pos-ible application of Nod factors for increasing pea productivity andymbiotic activity in general, including on compacted soils. Furthertudies are intended to assess these effects under field conditions.
. Concluding remarks
Symbiotic activity parameters (nitrogenase activity, total plantitrogen content and nodule mass) and shoot and root growtharameters of pea (at the flowering stage) grown in not compactedNC) and compacted (C) soil with and without Nod factors wereetermined. The results of this study showed the following:
1) Soil compaction significantly deteriorated all symbiotic androot and shoot growth parameters except of specific nitro-genase activity (per unit nodule weight) that was greater incompacted than not compacted soil.
2) Application of Nod factors significantly increased nitrogenaseactivity and total plant nitrogen content under NC and specificnitrogenase activity under both NC and C.
3) Irrespective of soil compaction and Nod factors nitrogenaseactivity was highly positively correlated with plant mass andplant N content.
ppendix A. Supplementary data
Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.apsoil.013.06.012.
eferences
bi-Ghanem, R., Carpenter-Boggs, L., Smith, J.L., 2011. Cultivar effects on nitrogenfixation in peas and lentils. Biol. Fertil. Soils 47, 115–120.
lmaraz, J.J., Zhou, X., Souleimanov, A., Smith, D., 2007. Gas exchange characteris-tics and dry matter accumulation of soybean treated with Nod factors. J. PlantPhysiol. 164, 1391–1393.
tti, S., Bonnell, R., Prasher, S., Smith, D.L., 2005. Response of soybean (Glycine max(l.) merr.) under chronic water deficit to LCO application during flowering andpod filling. Irrig. Drain. 54, 15–30.
otta, G.F., Tolon-Becerra, A., Lastra-Bravo, X., Tourn, M., 2010. Tillage and trafficeffects (planters and tractors) on soil compaction and soybean (Glycine max L.)yields in Argentinean pampas. Soil Till. Res. 110, 167–174.
uttery, B.R., Tan, C.S., Drury, C.F., Park, S.J., Armstrong, R.J., Park, K.Y., 1998. Theeffects of soil compaction, soil moisture and type on growth and nodulation ofsoybean and common bean. Can. J. Plant Sci. 78, 571–576.
akora, F.D., Atkins, C.A., 1991. Adaptation of nodulated soybean (Glycine max L.Merr.) to growth in rhizospheres containing nonambient pO2. Plant Physiol. 96,728–736.
ardanelli, M.S., de Córdoba, F.J.F., Estévez, J., Contreras, R., Cubo, M.T., Rodríguez-Carvajal, M.Á., Gil-Serrano, A.M., López-Baena, F.J., Bellogín, R., Manyani, H.,Ollero, F.J., Megías, M., 2012. Changes in flavonoids secreted by Phaseolus vulgarisroots in the presence of salt and the plant growth-promoting rhizobacteriumChryseobacterium balustinum. Appl. Soil Ecol. 57, 31–38.
’Haeze, W., Holsters, M., 2002. Nod factors structures, responses, and perceptionduring initiation of nodule development. Glycobiology 12, 79–105.
uzan, H.M., Mabood, F., Souleimanov, A., Smith, D.L., 2006. Nod Bj-V (C18:1,MeFuc) production by Bradyrhizobium japonicum (USDA110, 532C) at subop-timal growth temperatures. J. Plant Physiol. 163, 107–111.
uzan, H.M., Zhou, X., Souleimanov, A., Smith, D.L., 2004. Perception of Bradyrhizo-bium japonicum Nod factor by soybean [Glycine max (L.) Merr.] root hairs underabiotic stress conditions. J. Exp. Bot. 55, 2641–2646.
yachok, J.V., Tobin, A.E., Price, N.P.J., von Arnold, S., 2000. Rhizobial nod fac-tors stimulate somatic embryo development in Picea abies. Plant Cell Rep. 3,290–297.
gertsdotter, U., von Arnod, S., 1998. Development of somatic embryos in Norwayspruce. J. Exp. Bot. 49, 155–162.
logy 72 (2013) 181– 186
Gregorich, E.G., Lapen, D.R., Ma, B.-L., McLaughlin, N.B., VandenBygaart, A.J., 2011.Soil and crop response to varying levels of compaction, nitrogen fertilization,and clay content. Soil Sci. Soc. Am. J. 75, 1483–1492.
Guasch, L.M., de Felipe, M.R., Fernández-Pascual, M., 2001. Effects of different O2
concentrations on nitrogenase activity, respiration, and O2 diffusion resistancein Lupinus albus L. cv. Multolupa nodules. J. Plant Physiol. 158, 1395–1402.
Hamza, M.A., Anderson, W.K., 2005. Soil compaction in cropping systems. A reviewof the nature, causes and possible solutions. Soil Till. Res. 82, 121–145.
Hauggaard-Nielsen, H., Holdensen, L., Wulfsohn, D., Jensen, E.S., 2010. Spatial vari-ation of N2-fixation in field pea (Pisum sativum L.) at the field scale determinedby the 15N natural abundance method. Plant Soil 327, 167–184.
Hirsch, A.M., Lum, M.R., Downie, J.A., 2001. What makes the rhizobia–legume sym-biosis so special? Plant Physiol. 127, 1484–1492.
Horn, R., Way, T., Rostek, J., 2003. Effect of repeated tractor wheeling on stress/strainproperties and consequences on physical properties in structured arable soils.Soil Till. Res. 73, 101–106.
Khan, W., Prithiviraj, B., Smith, D.L., 2008. Nod factor [Nod Bj V (C18:1, MeFuc)] andlumichrome enhance photosynthesis and growth of corn and soybean. J. PlantPhysiol. 165, 1342–1351.
Kidaj, D., Wielbo, J., Skorupska, A., 2012. Nod factors stimulate seed germination andpromote growth and nodulation of pea and vetch under competitive conditions.Microbiol. Res. 167, 144–150.
Lindemann, W.C., Ham, G.E., Randall, G.W., 1982. Soil compaction effects on soybeannodulation, N2 (C2H4) fixation and seed yield. Agron. J. 74, 307–311.
Lipiec, J., Hatano, R., 2003. Quantification of compaction effect on soil physical prop-erties and crop growth. Geoderma 116, 107–136.
Lipiec, J., Horn, R., Pietrusiewicz, J., Siczek, A., 2012. Effects of soil compaction onroot elongation and anatomy of different cereal plant species. Soil Till. Res. 121,74–81.
Maj, D., Wielbo, J., Marek-Kozaczek, M., Martyniuk, S., Skorupska, A., 2009. Pre-treatment of clover seeds with Nod factors improve growth and nodulation ofTrifolium pretense. J. Chem. Ecol. 35, 479–487.
Maj, D., Wielbo, J., Marek-Kozaczuk, M., Skorupska, A., 2010. Response to flavonoidsas a factor influencing competitiveness and symbiotic activity of Rhizobium legu-minosarum. Microbiol. Res. 165, 50–60.
McKay, I.A., Djordjevic, M.A., 1993. Production and excretion of nod metabolites byRhizobium leguminosarum bv. trifolii are disrupted by the same environmen-tal factors that reduce nodulation in the field. Appl. Environ. Microbiol. 59,3385–3392.
Martyniuk, S., Oron, J., Martyniuk, M., 2005. Diversity and numbers of root-nodulebacteria (rhizobia) in Polish soils. Acta Soc. Bot. Pol. 74, 83–86.
Martyniuk, S., Wozniakowska, A., Martyniuk, M., Oron, J., 2000. A new sand pouch-plant infection technique for enumaration of rhizobia in soils. Acta Soc. Bot. Pol.69, 257–261.
Mathesius, U., Schlaman, H.R.M., Spaink, H.P., Sautter, C., Rolfe, B.G., Djordjevic, M.A.,1998. Auxin transport inhibition precedes root nodule formation in white cloverroots and is regulated by flavonoids and derivatives of chitin oligosaccharides.Plant J. 14, 23–34.
Miransari, M., Smith, D., 2009. Rhizobial lipo-chitooligosaccharides and gibberellinsenhance barley (Hordeum vulgare L.) seed germination. Biotechnology 8,270–275.
Mrema, A.F., Granhall, U., Sennerby-Forsse, L., 1997. Plant growth, leaf waterpotential, nitrogenase activity and nodule anatomy in Leucaena leucocephala asaffected by water stress and nitrogen availability. Trees 12, 42–48.
Nosalewicz, A., Nosalewicz, M., 2011. Effect of soil compaction on dehydrogenaseactivity in bulk soil and rhizosphere. Int. Agrophys. 25, 47–51.
Oláh, B., Brière, C., Bécard, G., Dénarié, J., Gough, C., 2005. Nod factors and a diffusiblefactor from arbuscular mycorrhizal fungi stimulate lateral root formation inMedicago truncatula via the DMI1/DMI2 signalling pathway. Plant J. 44, 195–207.
Pengthamkeerati, P., Motavalli, P.P., Kremer, R.J., 2011. Soil microbial activity andfunctional diversity changed by compaction, poultry litter and cropping in aclaypan soil. Appl. Soil Ecol. 48, 71–80.
Prithiviraj, B., Zhou, X., Souleimanov, A., Kahn, W.M., Smith, D.L., 2003. A host-specific bacteria-to-plant signal molecule (Nod factor) enhances germinationand early growth of diverse crop plants. Planta 216, 437–445.
Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Man-ual. Cold Spring Harbor, New York.
Schmidt, P.E., Broughton, W.J., Werner, D., 1994. Nod factors of Bradyrhizobiumjaponicum and Rhizobium sp. NGR234 induce flavonoid accumulation in soybeanroot exudate. Mol. Plant Microb. Interact. 7, 384–390.
Siczek, A., Frac, M., 2012. Soil microbial activity as influenced by compaction andstraw mulching. Int. Agrophys. 26, 65–69.
Siczek, A., Lipiec, J., 2011. Soybean nodulation and nitrogen fixation in response tosoil compaction and surface straw mulching. Soil Till. Res. 114, 50–56.
Souleimanov, A., Prithiviraj, B., Smith, D.L., 2002. The major Nod factor of Bradyrhi-zobium japonicum promotes early growth of soybean and corn. J. Exp. Bot. 53,1929–1934.
Voorhees, W.B., Carlson, V.A., Senst, C.G., 1976. Soybean nodulation as affected bywheel traffic. Agron. J. 68, 978–979.
Xie, Z.-P., Müller, J., Wiemken, A., Broughton, W.J., Boller, T., 1998. Nod factors andtri-iodobenzoic acid stimulate mycorrhizal colonization and affect carbohydratepartitioning in mycorrhizal roots of Lablab purpureus. New Phytol. 139, 361–366.
