CHEN Ding-an, WEI Xiao-wu, ZHANG Min, CHENG Wei, WANG Yu-shuang, LI Yi-lu, WU Shan-dong*, YI Hong-wei

1. Changde City Dingcheng District Agricultural Science Research Institute, Changde 415000, PRC；

2. Hunan Institute of Microbiology, Changsha 410009, PRC；

3. Hunan Double-Red Agro-Science Ecological Engineering Co., Ltd, Changsha 410205, PRC

Abstract In order to obtain high-efficiency organophosphorus solubilizing bacteria, 21 strains of organophosphate solubilizing bacteria were isolated from the rhizosphere soil of Camellia oleifera plants, and the transparent circle method was used for rescreening. Only 4 strains of bacteria could form transparent circle on organophosphorus medium. The D/d value of 4 strains of bacteria was between 1.62 and 2.71, among which the D/d value of strain Y6 was the highest (2.71). The available phosphorus content of the fermentation supernatant was 8.50～14.79 mg/L, which was 7.88～14.17 mg/L higher than that of CK. The strain Y6 had the highest soluble phosphorus content of 14.79 mg/L in the fermentation supernatant, which was 14.17 mg/L higher than that of CK. According to the colony morphology, physiological and biochemical characteristics and 16S rDNA sequence analysis of strain Y6, it is preliminarily determined that strain Y6 is Pseudomonas. Strain Y6 is beneficial to improve the supply of phosphorus in rhizosphere soil of Camellia oleifera and promote the growth of Camellia oleifera. It has great potential in the development of bio-organic fertilizer.

Key words Camellia oleifera； Organophosphorus solubilizing bacteria； Pseudomonas； Available phosphorus

1. Introduction

Phosphorus is one of the main nutrient elements for plant growth. The content of total phosphorus in farmland soil is high, whereas the content of available phosphorus is very low. This is because the phosphorus in the soil mainly exists in insoluble compounds, most of which cannot be directly absorbed and utilized by plants. In China, 74% of cultivated soil is deficient in phosphorus[1], and organic phosphorus accounts for 20%～50% of total phosphorus[2]. Generally, chemical phosphate fertilizer is used to provide phosphorus for plants, whereas the utilization rate of chemical phosphate fertilizer is very low, usually only 5%～25%[3]. Phosphate solubilizing bacteria can transform insoluble or slightly soluble phosphorus into plant available forms, and improve the utilization rate of phosphorus in soil. LIN Q Met al.[4]conducted research on the quantity and population structure of phosphorus solubilizing microorganisms in the rhizosphere soil of farmland, grass land, forest land and vegetable land, they found that the main organic phosphorus bacteria wasBacillus, followed byPseudomonas.

Camellia oleiferais one of the four unique woody oil-bearing trees in the world[5]. By the end of 2018, the total area ofCamellia oleiferaforest in Hunan Province has reached 1.407 4 million hm2, the total output of tea oil is 262 000 t, and the output value is 45 billion yuan, ranking first in China[6].Camelliaseed oil contains monounsaturated fatty acid—oleic acid, which can reduce the concentration of cholesterol and LDL in the blood and prevent cardiovascular disease[7]. The results show that there are phosphate solubilizing bacteria in the rhizosphere soil ofCamellia oleifera, which can improve the physical and chemical properties of the soil, promote the growth of plants, increase the yield of crops and be friendly to the ecological environment[8].

The research group collected samples from the rhizosphere soil of healthyCamellia oleiferaplants, isolated a batch of strains that can solubilize organic phosphorus, screened a strain of bacteria that can efficiently solubilize organic phosphorus, and analyzed its ability to solubilize phosphorus. Through the morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence analysis, we can preliminarily determine the classification status of strains, provide theoretical basis for the development of microbial fertilizer, so as to reduce the application amount of fertilizer, improve product quality and reduce production cost.

2. Materials and Methods

2.1. Test material

2.1.1. Sample collection

The rhizosphere soil of healthyCamellia oleiferaplants in Shaoyang City was collected and stored in a refrigerator at 4℃ after natural air drying in a cool place.

2.1.2. Culture medium

Organophosphorus medium: glucose 10 g, (NH4)2SO40.5 g, NaCl 0.3 g, KCl 0.3 g, MgSO4·7H2O 0.3 g, FeSO4·7H2O 0.03 g, MnSO4·4H2O 0.03 g, lecithin 0.2 g, CaCO35.0 g, yeast extract 0.4 g, agar 20 g, dd H2O 1 000 mL, pH value 7.0～7.5. LB medium refers to reference[9].

2.2. Test method

2.2.1. Isolation and purification of organophosphorus solubilizing bacteria

10 g of rhizosphere soil was weighed and added 90 mL of sterile water, and placed in shaking table at rotating speed 180 r/min for 30 min； after standing for 1 min, the suspension was diluted to 10-5according to the gradient, 100 μL was drawn from 10-3, 10-4and 10-5respectively and added to the plate of organic phosphorus culture medium, they were spread evenly, and cultured in a 30℃ incubator for 3～7 d； the single colony with transparent circle around was selected and purified for 3 to 5 generations. The pure strain was transplanted on LB slant culture medium and stored in refrigerator at 4℃ for standby.

2.2.2. Rescreening of organophosphorus solubilizing bacteria

Using the method of solubilizing phosphorus circle, the strains were spot connected to organophosph- orus medium plate. Each strain was cultured in 30 ℃ incubator for 4 d. The diameter of transparent circle (D) and the diameter of colony (d) were measured. According to the value of D/d, the phosphorus solubilizing ability of the strain was preliminarily determined.

2.2.3. Analysis of phosphorus solubilizing ability

The strains to be tested were inoculated in 50 mL liquid organic phosphorus medium, each strain was repeated three times, and the blank liquid organic phosphorus medium was used as control, 180 r/min, 30℃ constant temperature shaking table was used for 4 d. The culture medium was centrifuged at 4℃ for 10 000 r/min for 5 min, and the supernatant was diluted in appropriate times, then the content of available phosphorus was measured by molybdenum antimony anti-colorimetry method.

2.2.4. Identification of organophosphorus solubilizing bacteria

(1) Morphological identification of strains: the strains to be tested were inoculated on LB medium by scribing lines and cultured in a 30℃ incubator for 48 h. Colony shape, transparency, size, pigment, growth speed, edge characteristics,etc. were observed and recorded. Gram staining and optical microscopy were used. (2) Physiological and biochemical characteristics of the strain: identification shall be carried out according to the methods in theManual for Systematic Identification of Common Bacteria[10]. (3) Sequence analysis of 16S rDNA of the strain: according to the instructions of the kit, the DNA of the strain was extracted, and the PCR amplification of 16S rDNA was carried out with the general primer 27F/1492R. (4) PCR reaction conditions: initial denat- uration at 95℃ for 10 min； denaturation at 94℃for 30 s, annealing at 56℃ for 50 s, extension at 72℃ for 90 s, circulation for 30 times； extension at 72℃ for 10 min. The PCR products were detected by 1% agarose gel electroph- oresis. After being qualified, they were sent to the Shenggong Bioengineering (Shanghai) Limited by Share Ltd for sequencing. The sequences were compared with the GenBank data through the Blast program, and the sequences with high homology were selected to construct phylogenetic tree using MEGA 5.1 software to determine their taxonomic status.

2.2.5. The relationship between the content of available phosphorus and pH value in the fermentation broth of organophosphorus solubilizing bacteria

The strains to be tested were inoculated in organic phosphorus liquid medium after being activated in LB medium, and cultured in 180 r/min oscillation at 30℃, and then sampled at 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144 h respectively. The pH value of fermentation liquid was determined by acidimeter, and the available phosphorus content was determined by molybdenum antimony anticolorimetry method.

2.2.6. Determination of the growth curve of organophosphorus solubilizing bacteria

The strains to be tested were inoculated in LB liq- uid medium after being activated in LB medium plate. The volume of 500 mL triangular flask was 100 mL. The strains were cultured in 180 r/min constant temper- ature oscillation at 30℃, and the samples were taken every 2 h. The OD value of the fermentation solution at the wavelength of 600 nm was measured by UV spectrophotometer, and the growth curve was drawn.

2.2.7. Data processing

The test data were sorted out by Excel software and analyzed by SPSS 17.0 statistical analysis software.

3. Results and Analysis

3.1. Isolation and purification of organoph- osphorus solubilizing bacteria

21 strains with transparent circle were isolated from the plate of organophosphorus medium. They were named Y1 to Y21 respectively. After three generations of purification, the pure culture was inoculated on LB slant medium and stored in refrigerator at 4℃.

3.2. Rescreening of organophosphorus solubilizing bacteria

After rescreening 21 strains of bacteria, only Y3, Y6, Y11 and Y12 strains could form a clear circle on the organic phosphorus medium (Fig. 1). It can be seen from Table 1 that the D/d value of 4 strains of bacteria was between 1.62 and 2.71, among which the transparent circle diameter (D) of strain Y6 was 24.9 mm, the circle diameter (d) was 9.2 mm, and the D/d value was the largest, which was 2.71. The D/d values of the 4 strains were Y6＞Y12＞Y11＞Y3.

Table 1 Determination of phosphorus solubilizing capacity of 4 bacteria by transparent circle method

3.3. Analysis of the ability of organophosphorus solubilizing bacteria to solubilize phosphorus

After 4 strains of bacteria Y3, Y6, Y11 and Y12 were shaking cultured in constant temperature shaking table at 30℃ for 4 d, the content of available phosphorus in the supernatant was 10.37, 14.79, 8.50 and 9.49 mg/L, respectively, which were 9.75, 14.17, 7.88 and 8.87 mg/L higher than CK, and the increasing times were 12.71～22.85 times； the sequence of the available phosphorus content in the supernatant of the 4 strains was Y6＞Y3＞Y12＞Y11 from high to low, and the differences between the strains and between the strains and CK were significant. Among them, the available phosphorus content in the supernatant of strain Y6 was the highest, 14.79 mg/L, 23.85 times of CK. This strain was selected for the next test.

3.4. Identification of organophosphorus solub- ilizing bacteria

3.4.1. Morphological identification of strains

Strain Y6 was round colony on LB medium, white, no pigment, viscous. Microscopical observation showed that the bacteria was rod-shaped and did not produce spores, identified as gram negative bacteria.

3.4.2. Physiological and biochemical characteristics of strains

Strain Y6 can produce acid by glucose and xylose fermentation, mannitol and sucrose fermentation can't be used to produce acid. Y6 can not hydrolyze starch and use malonic acid, the result of V.P. test and methyl red test is negative. It can liquefy gelatin and the contact enzyme test is positive.3.4.3. 16S rDNA sequence analysis of the strain

The 16S rDNA sequence of strain Y6 was submitted to GenBank for homology retrieval. The homology between Y6 andPseudomonas arsenicoxydans(VC-1) was 99%. The strains with high similarity to the 16S rDNA sequence of Y6 were selected to establish phylogenetic tree by MEGA5.1 software adjacency method. The results were shown in Fig. 2. According to the colony morphological characteristics, physiological and biochemical characteristics of strain Y6 and 16S rDNA sequence analysis results, preliminary strain Y6 was identified asPseudomonas.

Fig. 2 Phylogenetic tree based on 16S rDNA sequence of strain Y6

3.5. The relationship between pH value and available phosphorus content of fermentation liquid of strain Y6

It can be seen from Fig. 3 that with the gradual decrease of pH value of fermentation liquid of strain Y6, the available phosphorus content of fermentation supernatant gradually increased. After 108 h of culture, the pH value of fermentation liquid was 6.4, and the available phosphorus content of fermentation supernatant reached the maximum value of 17.11 mg/L； with the increase of culture time, the pH value continued to decrease, and the content of available phosphorus in fermentation supernatant gradually decreased. After 144 h of culture, the pH value of fermentation supernatant was stable at 6.01, and the content of available phosphorus in fermentation supernatant was 13.96 mg/L. This indicated that the change of pH value of fermentation liquid of strain Y6 was not the necessary condition for organophosphorus solubilizing.

Fig. 3 Relationship between the content of available phos- phorus and pH value in fermentation broth of strain Y6

3.6. Growth curve of organophosphorus solub- ilizing bacteria Y6

The growth curve of microorganism can be divided into four stages: delay period, logarithm period, stability period and decline period. It can be seen from Fig. 4 that in LB liquid medium, the delay period of strain Y6 was within 4 h, after 4 h, the OD600value increased rapidly, and entered the logarithmic growth period. After 22 h, the OD600value reached the maximum, which was 5.21, and then entered the stable period, and remained there for quite a long time.

Fig. 4 Growth curve of strain Y6

4. Conclusion and Discussion

At present, the separation of organophosphorus solubilizing bacteria is mainly based on lecithin as a single source of organic phosphorus. The phosphatase secreted by organophosphorus solubilizing bacteria decomposes lecithin into choline, glycerin, fatty acid and phosphoric acid. Choline is further decomposed into amine, organic acid, carbon dioxide and alcohol, finally forming a transparent circle on the organic phosphorus medium[11]. The research group sampled the rhizosphere soil of the healthyCamellia oleiferaplants, and totally 21 strains with transparent circles on the organic phosphorus medium were isolated. After rescreening with the method of solubilizing phosphorus circles, only 4 strains of bacteria could form obvious transparent circles on the organic phosphorus medium, and the other 17 strains had no transparent circles and lost the phenomenon of phosphorus solubilizing. VENKATESWARLU Bet al.[12]also reported that most phosphate solubilizing bacteria will lose their phosphate solubilizing ability after several passages.

The D/d value of 4 strains of bacteria was between 1.62 and 2.71 in 30℃ incubator for 4 d, among which the transparent circle diameter (D) of strain Y6 was 24.9 mm, the circle diameter (d) was 9.2 mm, and the D/d value was the largest of 2.71. The value of D/d ratio only showed that bacteria had the ability of phosphate solubilizing, and the ability of phosphate solubilizing could not be determined. Many scholars had similar views[13-14]. 4 strains of bacteria were cultured in a constant temperature shaking table at 30℃ for 4 d, the content of available phosphorus in the supernatant of fermentation was 8.50～14.79 mg/L, the content of available phosphorus in the supernatant of CK was 0.62 mg/L, the increase of available phosphorus in the supernatant was 7.88～14.17 mg/L, and the content of available phosphorus in the supernatant of strain Y6 was the highest, 14.79 mg/L, 14.17 mg/L higher than CK. The order of D/d value of 4 strains of bacteria was Y6＞Y12＞Y11＞ Y3 from the largest to the smallest, while the order of available phosphorus content of the fermentation supernatant of 4 strains was Y6＞Y3＞Y12＞Y11 from the highest to the lowest, which indicated that there was not always a positive correlation between D/d value of phosphate solubilizing bacteria and available phosphorus content of the fermentation supernatant, which was consistent with the research results of WU S Det al.[15].

The pH value of fermentation broth of strain Y6 decreased with the increase of culture time, and the pH value of fermentation broth was stable at 6.01 after 144 h of culture. The content of available phosphorus in fermentation supernatant increased first and then decreased. After 108 h of culture, the content of available phosphorus in fermentation supernatant reached the maximum value, which was 17.11 mg/L, indicating that the change of pH value of fermentation broth of strain Y6 was not the necessary condition for organophosphorus solubilizing. ZHAO X Ret al.[16]had shown that there was a certain correlation between the available phosphorus content of fermentation broth and the pH value of culture broth, whereas the decline of pH value of culture broth was not a necessary condition for phosphorus solubilizing. For this reason, many scholars also found that the correlation between the two was weak[17-18]. The decrease of pH value of fermentation medium is related to the secretion of organic acids by the phosphorus solubilizing bacteria. There are other sources of H+, such asexchange mechanism and respiration. At present, it is considered that phosphatase secreted by organophosphorus bacteria is one of the main mechanisms of phosphorus solubilization. ZHANG S H[19]thinks that the organic acids, proteins and polysaccharides secreted by organophosphorus bacteria may also have a certain effect on phosphorus solubilization.

According to the colony morphology, physio- logical and biochemical characteristics and 16S rDNA sequence analysis of strain Y6, it is preliminarily determined that strain Y6 isPseudomonas.