Enhancement of nodulation and plant growth in dry bean by inoculations with different microorganisms

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Selçuk University, Faculty of Engineering, Environmental Engineering Campus, 42130 Selcuklu, Konya, Turkey

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(USDA 9032), two genetically modified strains, which originated from $!
 , and a plant growth-promoting-rhizobacterium, $!
 Sp7 either alone or in com-bination with the strain 9032 were inoculated into dry bean ( !
"! L.) to determine which inoculum yield the highest nodulation and plant growth. The experimental design was completely randomized design with 6 replications (pots) per treatment. Double inoculation with the strains Sp7 and 9032 yielded the highest nodule number and weight. While Sp7 significantly increased nodule number, it did not significantly increase nodule weight compared to the control. There was not a sta-tistically significant difference among the treatment means in terms of nitrogen content. However, a ge-netically modified $! and the strain Sp7 sig-nificantly increased accumulations of nitrogen in shoots and whole plants, compared to the control. Consequently, the enhancement of nodulation does not always lead to higher plant nitrogen contents. In case of $!, there may be some additional explanation(s) (e.g. improved plant root system) for the increase in plant growth.

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 , genetically modified microorganism, $!
, nodulation, !
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Legumes assimilate ammonium (NH4+), a product from the process of nitrogen (N2) fixation, which is mediated by a common soil bacterium,  $!, which infects the plant root cells, forming nodules, the small outgrowths of the roots. Unfortu-nately, dry bean ( !
"!
L.) can benefit little from N2-fixation because it usually has a short vegetative life, hence there is little time for nodule formation (nodulation) and N2-fixation [1, 2, 3]. Fur-thermore, dry bean is usually grown in marginal con-ditions such as acidic soils and hot climates, leading to relatively less photosynthesis products that can be allocated for N2-fixation [4, 5]. Some genotypic and

phenotypic parameters for different bean genotypes were previously determined [6]. The effects of ferti-lization with sulphur and potassium on nitrogenase and microbial activities in soil, in which broad bean (

) was grown was determined [7]

Some studies have shown that genetically mod-ified rhizobia can increase nodulation or nitrogen fixation. For example, chromosomal integration of a gene, which encodes for a short spectrum antibiotic, trifolitoxin (TFX) was determined [8]. TFX produc-tion enhanced the ability of the transgenic microor-ganism to colonize rhizosphere and induce nodula-tion. The TFX-producing strain was later proven to perform better in nodulation competitiveness, com-pared to the near isogenic non-TFX-producing strain under field conditions [9]. On the other hand, a  $!
  cytochrome mutant increased symbiotic nitrogen accumulation in dry bean [10]. There are no data, which compare the TFX-producing strain and the cytochrome mutant in nitrogen and dry mass ac-cumulations.

$!, a plant-growth-promoting rhizo-bacterium was shown to enhance nodulation
[11,12]; acetylene reduction activity [10]; and some plant growth parameters [11,12,13] in dry bean. $ !
causes earlier nodule formation and it increases susceptibility of roots to nodulation. However, there is no report on how $! or $! $!
inoculations compare the inoculation of genetically modified rhizobial strains, namely the TFX-producer (TFX) and the cytochrome mutant (CFN030). Consequently, the objective of this study is to compare the wild type $!
 , the ge-netically modified rhizobial strains, and $ ! (either single or double inoculated with $ !
 ) in nitrogen and dry matter accumulation. " !

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The microorganisms used

in this study are listed in Table 1. The wild type and genetically modified $!
  strains were cul-tured in yeast mannitol agar (YMA) at 28 o_{C for 72} h. The agar contained appropriate antibiotics for the

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strains TFX and CFN030
[9, 10]. The cells were scraped out to a sterile saline solution (8.5 g NaCl 1000 ml-1_{) aseptically. $! was cultured in} a malate minimal liquid medium supplemented with mineral N [14]. The cells in the liquid culture were harvested by two times centrifugation for 10 min at 1,000 ×g in the sterile saline solution. The population densities in the microbial suspensions were adjusted to 106 _{CFU ml}-1 _{by dilution with the sterile saline} so-lution after determining the optical density of each suspension at a spectrophotometer. Similar to $!,  !
! was previ-ously grown in a similar medium [15].

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treatments in this study were () Control (heat killed bacteria); () $!
 
USDA 9032 (wild type); () $!
 
(TFX-producer); (") $!
 
(CFN030); (") $!

Sp7; (") $!
 
USDA 9032 + 

Sp7. The experimental design was completely randomized de-sign with 6 replications per treatment. The soil used was taken from a field that has been cultivated for crops including dry bean. It was classified as Fine-loamy, mesic, typical Ustifluvent. Some soil proper-ties determined by routine soil analysis methods are as follows: Organic matter, 2.5 (%); pH, 7.85; EC, 1.49 (dS m-1_{); CaCO}

3, 11.2 (%); Total N, 0.05 (%); Olsen-P, 15 mg kg-1_{; Texture, Clay loam. The soil} was sieved thorough 2 mm sieve, heat sterilized by 24 hour in a chamber, and loosely packed up into the pots which had been surface sterilized with 10 % bleach.

Dry bean ( !
"! L.) seeds were surface sterilized by dipping them into solutions, which contained ethyl alcohol (95 %) for one minute and 1 % sodium hypochloride (NaOCl) for two minutes afterwards. The seeds were washed six times in sterile water to remove traces of sodium hy-pochloride. They were germinated in sterile agar (1.5 %) in dark at 30 o_{C for 3 days. The seedlings were} sown into the pots containing the sterile soil, as one seedling per pot. The plants were watered as needed. The plants were removed from the pots gently at 45 days after inoculation (DAI), in such a manner to minimize the losses of roots and nodules. The rec-orded data include nodule numbers and weight per plant; root and shoot dry weights; plant height. Ni-trogen content of samples from root and shoot were

determined by Kjeldahl method.

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performed by SPSS (V 10.0). The treatment effects were partitioned by one way-ANOVA. The signifi-cant differences among the treatment means were de-termined by Duncan’s Multiple Range Test.

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The number of nodules per plant significantly increased (ANOVA; =4.19; =5; =0.005) from 11 in the control to 196 in the wild type $! (Figure 1a). The $! co-inoculation did not significantly change the number of nodules. $ ! alone inoculation did not significantly en-hance nodule numbers compared to the control. The nodule weight per plant significantly increased (ANOVA; =6.93; =5; =0.0002) from 27 mg in the control to 704 mg in the wild type strain (Figure 1b). The $! co-inoculation further in-creased this figure to 885 mg, however, the increase obtained by the $! co-inoculation was not significant at 5 % significance level.

The wild type $! inoculation yielded a shoot mass of 5.72 g plant-1_{, which is significantly} higher (ANOVA; =4.00; =5; =0.007) than 4.53 g plant-1 _{in the control (Figure 1c). The total dry} mat-ter accumulations in the genetically modified strains are statistically similar to the one in the control (Fig-ure 1d). These fig(Fig-ures are in agreement with the nod-ulation data. However, $! alone inocula-tion significantly increased total dry matter accumu-lation (ANOVA; =3.72; =5; =0.01), although it relatively yielded much less numbers of nodule than the wild type strain (Figure 1a). The shoot to root ra-tio (S:R) did not significantly change among the treatments (Data are not shown). The plants inocu-lated with the strain TFX and $! were non-significantly higher than the control plants (Figure 1e). However, the TFX and $! treatments significantly increased total nitrogen accumulation in the shoots (ANOVA; =3.92;
=5; =0.008) and the whole plants (ANOVA; =3.48; =5; =0.015) compared to the control (Table 2).

Strains Characteristics Source or reference

$!
  USDA 9032 (wild type) P. V. Berkum(1)


 
TFX pT2TFXK (pTR102 with # , Robleto et al., 1997

Kmr_{) transferred CE3}(2)


 
CFN030 Azr _{Tn5-mob mutant of CE3 Miranda et al., 1996}

$!
 Strain Sp7 W. Zimmer(3)

1 _{USDA/ARS Beltsville $! Germplasm Collection, Beltsville, MD.}

2 _Smr _{derivative of $!
  CFN42.}

3 _{Universitat Köln, Gyrohofstr, FRG.}

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Treatment % N Total N-Accumulation (mg/plant)

Shoot Root Whole _{Shoot Root} Whole

Control 2,17 1,73 2,08 _{97 (a)} 19,8 117 (a)

9032 2,22 1,76 2,12 _{109 (a)} 29,5 136 (ab)

TFX 2,50 1,79 2,36 _{143 (b)} 26,3 170 (b)

CFN030 2,09 1,77 2,02 _{96 (a)} 26,0 122 (a)

Sp7 2,24 1,81 2,20 _{143 (b)} 29,4 169 (b)

9032 + Sp7 2,14 1,74 2,06 _{114 (ab)} 23,6 138 (ab)

† _{The numbers represent the mean of six replications. The means followed by the same letter are not significantly}

different at 5 % significance level as per the Duncan’s Multiple Range Test.

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Some of the local rhizobial strains probably are resistant to our heat killing procedure since small numbers of nodule were formed in the control treat-ment. However, most of the nodules probably were formed by the inoculant rhizobia in the bacterial treatments since the number of nodules formed in the control was too low. For example, the ratio of the number of nodules formed in the control to the num-ber of nodules formed in the strain 9032 was 5.6 : 100.

The number and weight of nodules formed by the strain TFX is lower than the ones formed by the other inoculations (Figures 1a and b), which did not yield significant improvement in symbiotic nitrogen accumulation compared to the control (Table 2). Our results are confined only to one set of experiment. It is also noteworthy mentioning that not only the TFX but also the genetically modified CFN030 produced less numbers of nodules than the wild type strain. This is especially important in the case of the strain TFX, which was proven to out-compete its near iso-genic non-TFX-producing counterpart in both sterile and non-sterile conditions [8]. Only the proportion of the nodules formed by the TFX-producing or non-producing strains to those formed by the TFX-sensitive strain was compared, with almost no atten-tion being paid to the total number of nodule or sym-biotically fixed N2 [8]. The genetic modifications could reduce the overall fitness of the microorgan-ism.

$! plays role in the root development and mineral accumulation in plants. However, we did not determine a significant difference in root mass among the $! inoculation and the control (Data are not given). $! inocula-tion could have increased accumulainocula-tion of nitrogen through improved root system. It was reviewed that $! enhances the numbers and length of the lateral roots and root hairs, which in turn can result in improved plant nutrient uptake [16]. There was not a significant improvement in root mass by $! inoculation in the current study. How-ever, it should be noted that the structural improve-ment in the root system (e.g. increase in root surface

area) does not always have to result in higher root biomass.

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The investigation reported in this paper (BAP-06701427) is in connection with a project of Selçuk University.

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[3]
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"!
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"!
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[9]
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[10]
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"! L
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[13]
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"! L.) as influenced by $! and $! inoculants. En-viron Ecol. 11, 581-583.

[14]
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[15]
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Bashan, Y., Holguin, G. (1997)

Azospirillum-plant relationships: environmental and physio-logical advances. Can J Microbiol. 43, 103-121

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Selçuk University, Faculty of Engineering

Environmental Engineering Campus, 42130 Selçuklu, Konya – Turkey