Influence of two phosphate-potassium solubilizing bacterial species on biomass and nitrate concentration on mustard greens (Brassica juncea (L.) Czernjaew) cultivated on acid sulfate soils

Two field trials were conducted to determine the effect of P-K solubilizing bacteria and nitrogen chemical fertilizer on growth, yield and nitrate in leaf of mustard greens Brassica juncea L.) cultivated on acid sulfate soils. Eight P-K solubilizing bacterial strains composed of 3 strains of Acinetobacter calcoaceticus and 5 strains of Rhizobium sp., bacterial liquid were directly watered into plant at 3 stages [6, 12 and 21 days after planting] during vegetable cultivation, chemical fertilizer (60N – 40 P2O5 – 20 K2O) and control (no-inoculation). The study revealed that eight P-K solubilizing bacterial strains have good characteristics as nitrogen fixation, phosphate and potassium solubilisation. Application of nitrogen chemical concentration, increasing nitrate concentration of leaf of mustard greens. Application of bacterial liquid with Acinetobacter calcoaceticus strain NT4 and Acinetobacter calcoaceticus strain NT30 strain, Rhizobium tropici strain N18 and Rhizobium leguminosarum strain K35 on mustard greens cultivated on acid sulfate soils (basal fertilizer: 40 P2O5 – 20 K2O) was the best cultural practice because the this model not only supported the highest biomass, but also decreased nitrate concentration in leaf of mustard greens.

The aims of this study (i) study on three phosphate and potassium solubilizing bacteria strains belonged to Acinetobacter calcoaceticus and five strains of Rhizobium sp…..on mustard greens (Brassica juncea (L.) Czernjaew) cultivated on acid sulfate soils and (ii) stimulation of nitrate concentration in leaf of mustard greens.

Soil
Chemical characteristics of soil used in this study were presented in Table 1. These results were analyzed in Advanced Lab., Can Tho University, Vietnam.

Bacterial strains
Eight strains were chosen to study (from the result of Nguyen Thi Don and Cao Ngoc Diep' experiment) [4] composing of three Acinetobacer calcoaceticus strains (NT1, NT4, NT30) and five Rhizobium strains (Rhizobium tropici CA29, Rhizobium. leguminosarum K35, Rhizobium. leguminosarum DG1, Rhizobium sp. Tu09, Rhizobium. tropici N18). All eight strains were isolated from weathering-rock samples from Cam Mountain, An Giang province and selected many times and they are the high potential strains in bio-fertilizer production.
Three Acinetobacter calcoaceticus strains NT1, NT4, NT30, and five Rhizobium sp. strains CA29, DG1, TU9, N18, K35) were each proliferated by incubation in container 120-L with 100 liters water of 10% sugar in 10 days. In this stage, bacterial density reached 10 7 cells/ml and then they already used for inoculating (by mixed bacterial liquid with water and watering for mustard greens) at 6, 12 and 21 days after planting.

Composting procedure
Compost was prepared from rice straw (Oryza sativa). The compost added with 0.02% Trichoderma spore (Dept. of Plant Protection, College of Agriculture, Can Tho University), incubated by covering plastic membrane; the compost was inverted and watering fortnightly. After 6 weeks, the volume of compost was reduced 50%, compost to keep moisture at 50 -60%, compost was incubated 4 weeks and compost from the bucket, air dried and later sieved to remove the shaft, shredded and bagged. Compost used in this study with pH and physical and chemical characteristics presented as follows: pH 6 Superphosphate and KCl was mixed and spread evenly 1 day before seedlings put into the holes in every plot unless control treatment; urea (divided 2 times to apply mustard greens) was mixed into water to watering to mustard greens at 4 DAP (50%) and 12 DAP (50%).

Experimental procedures
Experiments were conducted in greenhouse conditions with four blocks, consisting of nine beds per plot. Each beds was size of 1 x 1 m and the block was size of 13 x 5.5 m. The total land area was 71.5 m 2 ( Figure 1). The seedlings were prepared in the plastic glass with one seed per glass ( Figure 2). The experimental design was a randomized complete block design with three replications.  Insecticides did not used in the experiment. Weed was controlled by hand. Leaf plants were harvested at 28 days to measure plant height, leaf length, leaf number/plant, weight of a plant, biomass yield, available ratio and nitrate content in mustard greens leaf.

Results and discussion
In general, mustard greens seedlings developed very well on acid sulfate soil in the first day because they were planted in compost (in plastic cup) ( Figure 2). Furthermore, their roots only developed from 5 -7 cm depth and roots did not grow into the acid sulfate soil.

Effects of NPK and bacterial liquid on plant height and yield component of mustard greens
Application of chemical fertilizer (NPK) and two species (phosphate and potassium solubilizing bacteria) increased plant height of two vegetables and plant height was the lowest in the control treatment, especially application of Acinetobacter calcoaceticus strain NT4 (NT4 treatment) and Acinetobacter calcoaceticus strain NT30 (NT5 treatment) enhanced 4 parameters of yield components of mustard greens while only treatment of Rhizobium tropici strain N18 + 40 P2O5-20 K2O (NT6 treatment) increased 4 parameters of yield components of mustard greens therefore Rhizobium tropici strain N18 had the good ability of nitrogen fixation (replaced or saved of 60 kg N/ha). Thus this strain provided enough of nutrients for the growth of mustard greens in the short time (4 weeks).

Figure 3
Effects of NPK and 3 strains of Acinetobacter sp. on biomass (ton/ha) and trade productivity (ton/ha) of mustard greens cultivated on acid sulfate soils The numbers followed by the same letter do not differ at 1% level significantly This result led to biomass and trade productivity of treatment of Acinetobacter calcoaceticus strain NT4 + 40P2O5 -20K2O (treatment NT4) and Acinetobacter calcoaceticus strain NT30 + 40P2O5 -20K2O (treatment NT5) had the highest and differed from other treatments ( Figure 3). In experiment with Rhizobium sp. as Rhizobium tropici strain N18 + 40P2O5 -20K2O (treatment NT6) had the highest biomass and trade productivity and it differed from others ( Figure 4). Therefore, in genus Acinetobacter and Rhizobium had the strains the famous strains as they can fix nitrogen from the air to provide for the growth of mustard greens beside they dissolve insoluble phosphate and potassium. Yield component of 2 strains (Acinetobacter calcoaaceticus and Rhizobium sp.) correlated with biomass very closely (**) ( Table 2). In both experiments, application of Acinetobacter calcoaceticus strain NT1 + 40P2O5 -20K2O (NT3 treatment) and 60N -40P2O5 -20K2O treatment (NT2 treatment) did not differ from biomass but two treatments: NT4 treatment (Acinetobacter calcoaceticus strain NT4 + 40P2O5 -20K2O) and NT5 treatment (Acinetobacter calcoaceticu strain NT30 + 40P2O5 -20K2O) had the highest biomass and differed from others significantly. Two strains of genus Acinetobacter (Acinetobacter calcoaceticus NT4 and Acinetobacter calcoaceticus NT30), therefore strain NT4 and strain NT30 saved 60 kg N/ha or 60 kg N/ha from nitrogen fixation and this amount of nitrogen provided directly to mustard greens through biological nitrogen fixation.
Similarly, application of Rhizobium tropici strain CA29 + 40P2O5 -20K2O (treatment NT3) and Rhizobium sp. strain TU09 + 40P2O5 -20K2O (treatment NT5) and 60N-40P2O5-20K2O (treatment NT2) did not differ from biomass but two treatments (treatment NT7) Rhizobium tropici strain N18 + 40P2O5 -20K2O and Rhizobium leguminosarum strain K35 + 40P2O5 -20K2O (treatment NT5) had the highest biomass and they differed from others significantly. However with treatment of Rhizobium sp. strain DG1 + 40P2O5 -20K2O (NT6 treatment) had biomass lower than 60N-40P2O5-20K2O treatment (NT2 treatment) and its biomass only was higher than control (no fertilizer, no bacteria). Both genus (Acinetobacter and Rhizobium sp.) or these strains as Acinetobacter calcoaceticus NT4 and NT30 strains and Rhizobium tropici strain N18 and Rhizobium leguminosarum strain K35 were strains not only good P-K solubilisation but also strong nitrogen fixation in the short time to provided nutrients (NPK) for the growth of mustard greens cultivated on acid sulfate soils. Besides, application of inorganic nitrogen in mustard greens cultivation enhanced nitrate concentration in leaf with high levels (>650 mg/kg) while treatments of bacterial liquid in mustard greens cultivation did not increase nitrate concentration in leaf (equivalent with control) ( Figure 5 and Figure 6).
The numbers followed by the same letter do not differ at 1% level significantly In 1991, Martinez-Romero and colleagues [5] identified a new species of common bean symbionts: Rhizobium tropici. This new rhizobial species was described as aerobic, Gram-negative, with optimal pH for growth ranging between 5 and 7, and characterized by high genetic stability of the symbiotic plasmid and tolerance to tropical environmental stresses such as high temperature and low soil pH; it can nodulate in Phaseolus vulgaris (common bean) and some other leguminous species [6] and it was recognized as an effective symbiont of the common bean, Leucaena leucocephala and some other leguminous species [7]. Rhizobium tropici had good competition with other rhizobia during successive bean cultures and this strain is currently recommended (authorized) for the production of commercial rhizobial inoculant for common bean production in Brazil [8].
The first 'Acinetobacter' microorganisms were identified in 1911 by the Dutch microbiologist, Beijerinck, who assigned the bacteria the name Micrococcus calcoaceticus following their isolation from soil [9]. Acinetobacter cells are Gramnegative short rods (coccobacilli), measuring 1.0-1.5 by 1.5-2.5 microns during growth; they often become more coccoid during the stationary phase. The Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex has emerged as a high priority among hospital-acquired pathogens in intensive care units (ICUs), posing a challenge to infection management practices [9]. However, Acinetobacter sp and A. calcoaceticus play an important role in the fermentation process of the cocoa bean. For example, bacteria from the genus Acinetobacter oxidize ethanol, produced to form acetic acid. While acetic acid the acetic acid is formed during the cocoa fermentation, the temperature increases due to biochemical reactions, killing germ and breaking the cell-wall [10].
Kudpeng et al. [11] recognized only M. caseolyticus and Acinetobacter calcoaceticus were capable of gold bioleaching and the growth supernatant of M. caseolyticus and A. calcoaceticus can be applied in gold bioleaching. Seventy-six isolates were isolated from 25 rhizosphere soil samples of 13 different leaf-eating vegetables species grown in 6 districts of Can Tho City. Among them, 48 isolates had good characteristics as nitrogen fixation, phosphorus solubility and IAA synthesis. Especially, one of good PGPR was identified as Acetinetobacter calcoaceticus [12]. The good P-K solubilizing bacteria isolated in granite-weathered materials from CAM mountain were Acinetobacter calcoaceticus [13]. Therefore, among Aciinetobacter calcoaceticus isolates influenced to human healthy, there were Acinetobacter calcoaceticus isolates as PGPR to bio-fertilizer production for crops cultivation as in the our experiment [4].
Twelve P-K solubilizing bacterial strains without nitrogen fertilizer had higher biomass than 60 kg N/ha treatment but the Acinetobacter calcoaceticus NT4 and Acinetobacter calcoaceticus NT30 strains not only had high biomass but also had stable soil fertility through high pH soil, N total soil and OM in comparison to initial [4]. Furthermore, Acinetobacter calcoaceticus strain NT4 and strain NT30 were the best strains because they not only supported the highest biomass, low nitrate residue in leaf but also improved soil fertility through pH soil, N total, organic matter after harvesting vegetable for mustard greens cultivation in acid sulfate soil. Besides, Rhizobium tropici N18 strain together with Acinetobacter calcoaceticus NT4 and NT30 strains were the best strains for mustard greens cultivation in acid sulfate soils [4]. In this experiment, these strains (NT4, NT30 and N18 strains) demonstrated the good characteristics as good phosphate-potassium solubilisation and N fixation provided to mustard greens in short time.
However increasing nitrogen chemical concentration from 25N up to 50N, enhanced nitrate in leaf of mustard greens, especially 3 strains (NT30, N18, K35) had nitrate in leaf exceeded over 500 mg/kg [4] but in this experiment, application of bacterial strains without nitrogen fertilizer led to nitrate concentration in leaf of mustard greens as equivalent as control treatment ( Figure 5 and Figure 6),, this showed that application of inorganic nitrogen fertilizer for mustard greens cultivation stimulated and enhanced nitrate concentration in leaf of mustard greens, this result also demonstrated by Nguyen Thi Lan Huong [14] when she studied on the relationship of 8 kinds of vegetables with nitrogen fertilizer at Kim Boi market, Ha Noi city. Ministry of Agriculture and Rural Development, Vietnam promulgated with Decision: 99/2008/QĐ-BNN 15/10/2008 limitation of nitrate in leaf of vegetable are 500 mg/kg. Therefore vegetables production, especially leaf-eating vegetable cultivation not only supported high biomass and good quality but also improved soil fertility and environmental protection.
The strains of phosphate-potassium solubilizing bacteria had the characteristics as Plant growth-promoting rhizobacteria (PGPR) benefit plants through different mechanisms of action, including, for example, (i) the production of secondary metabolites such as antibiotics, cyanide, and hormonelike substances; (ii) the production of siderophores; (iii) antagonism to soilborne root pathogens; (iv) phosphate solubilisation; and (v) dinitrogen fixation [15] when they studied on Rhizobium and Bradyrhizobium species as plant growth promoting rhizobacteria on nonlegumes: on radishes (Raphanus sativus L.). With these results, we will select the good strains to produce bio-fertilizer for vegetable cultivation, especially on acid sulfate soils.

Conclusion
Four P-K solubilizing bacterial strains (Acinetobacter calcoaceticus N4 and NT30) and (Rhizobioum tropici N18 and Rhizobioum leguminosarum K35) supported the growth of mustard greens (biomass) but nitrate concentration in leaf of mustard greens (Brassica juncea) had the lowest; application of nitrogen fertilizer enhanced nitrate concentration in leaf of mustard greens but using these bacterial strains increased not only biomass but also decreased nitrate concentrate in leaf of mustard greens; these strains will be suggested to use in bio-fertilizer production for leaf-eating vegetable nearly in the future.