Uncle Hu
Editor|Uncle Hu
preface
Phosphorus is an important material basis for the survival of organisms, and it is also a pervasive nutrient constraint.
Its proportion in crops is second only to N and K, however, most of the world's arable land has a phosphorus deficiency problem.
Of the world's 1.319 billion hm2 of land, nearly 43% of the land has phosphorus deficiency, while nearly two-thirds of China's land has soil phosphorus deficiency.
At present, there is a general lack of effective phosphate fertilizer in mainland agricultural production, and how to effectively promote agricultural production is a scientific problem that needs to be solved urgently.
Agricultural production should be improved from the following three perspectives: First, the application of phosphate fertilizer to increase phosphorus in agricultural production.
The second is to use phosphorus-dissolving bacteria as raw materials, and through the research of phosphorus-dissolving bacteria, it lays a solid theoretical and practical foundation for the development of phosphorus-dissolving fertilizers. The third is how to cultivate new plant varieties with high phosphorus absorption and utilization of phosphorus in soil.
Although the application of phosphate fertilizer can in a certain sense improve the lack of efficient phosphorus in the soil of the vast area of the mainland, after a large amount of phosphate fertilizer enters the farmland, it will change to calcified and closed storage at a faster rate.
At the same time, with the continuous demand for phosphate fertilizer, the existing phosphate ore is also becoming more and more scarce.
The implementation of this project has important theoretical and practical value for further improving the utilization rate of phosphate fertilizer in mainland farmland, developing high-quality phosphorus-dissolving bacteria, and selecting and breeding new varieties of phosphorus-efficient crops.
Soil microorganisms are the largest, richest and most nutritious class of organisms, which can convert organic phosphorus into inorganic phosphorus that can be used by crops, and are called "phosphorus-dissolving bacteria".
Phosphorus dissolving bacteria are not only diverse, but also have a large number in nature, which is a resource worth developing and studying. IN 1903, STALSTROM ET AL. FIRST REPORTED PHOSPHORUS-SOLUBLE BACTERIA, FUNGI, ACTINOMYCETES, AND CYANOBACTERIA.
Globally, the earliest and most widely used phosphorus-dissolving microorganism is Bacillus megasiella. In subsequent studies, various bacillus, pseudomonas and some bacilli and some fungi were also developed as fungal fertilizers, which were used extensively in the production of biofertilizers.
The Department of Resources and Environment of Nanjing Agricultural University selected and bred a transgenic rice with extremely high phosphorus efficiency, OsPT4, which converted a phosphorus transporter with high affinity of OsPT4 into the recipient body to obtain transgenic rice with high phosphorus efficiency.
OsPT4 is a class of phosphorus transporters with strong biological activity, which can promote the uptake and utilization of phosphorus by plants. Based on the above results, can the development of phosphorus-dissolving bacteria promote the hydrolysis of organic phosphorus in soil, and can its utilization rate of phosphorus be improved?
Therefore, in this project, OsPT4 was used to screen out a bacterium that can effectively degrade organophosphorus from the rhizosphere soil layer of OspT4.
Firstly, the degradation performance and dissolved phosphorus characteristics of the bacteria were analyzed, and then the bacteria were compared with the phosphorus-dissolving bacteria obtained in conventional farmland to determine the application potential of the bacteria in plants.
Materials and methods
The continuous cropping test of phosphorus efficient genotype is planned to be conducted in 2014 at the Eco-Environmental Science Observatory of the Chinese Academy of Sciences.
First, put a 1.0 g rhizosphere soil sample into 9 mL of sterilized water, mix in vortex for 30 seconds, and leave for 10 minutes.
The 1mL bacterial solution that has been cultured is coated on Monkina's solid medium in the order of 10-1~10-6, each medium is 3 types.
After 5 days of constant temperature incubation at 30 °C, 10-5 is the appropriate concentration. According to the above method and the optimal dilution factor, organophosphorus bacteria were isolated from the soil sample.
A single bacterium that can generate a phosphorus ring was selected, purified by scribing for 3~5 passages, so as to obtain pure bacteria, and put them into LB medium for amplification, and then stored by glycerol method.
Monkina solid and organophosphate was used as the strain, and the colonies were initially identified for 3 days through morphological analysis and according to the contents of the Manual for Systematic Identification of Common Bacteria.
By measuring the community size and phosphorus ring size of bacteria, the solubility index of bacteria was obtained, and the phosphorus dissolving performance of bacteria was preliminarily evaluated. The solubility is calculated as follows: solubility index = diameter of the dissolved phosphorus ring / colony diameter.
Results & Analysis
Fifteen degradable organophosphorus detoxification bacteria were screened from the rhizosphere soil of OsPT4 using Monkina solid organophosphorus fermentation broth. Through three generations of purification and fermentation, 6 strains of bacteria with good phosphorus-releasing properties and stability were obtained (Figure 1B).
The morphology, size and color of six organophosphorus bacteria were observed and recorded, and the analysis of six organophosphorus bacteria showed that all six organophosphorus bacteria were gram-negative (Table 1).
The phosphorus dissolving performance of bacteria is usually evaluated by solubility, 24-hour phosphorus dissolving rate and other indicators.
From the solubility index, the phosphorus-dissolving ability of the six organophosphorus-solving strains was different, and the order from largest to smallest was: Y7>Y11>Y1>Y13>Y4>Y6, of which the soluble index of Y7 strain was 6.33 and Y11 was 3.81 (Table 2).
In addition, the phosphorus-dissolving ability of six organophosphorus strains was quantitatively studied, and it was found that the concentration of inorganic phosphorus in Y7 after 24 h of culture was 87.43mgL (Figure 1 Fig. 2), which was significantly higher than that of the other five strains.
After 24 hours of incubation, the pH of each bacterial solution decreased significantly and showed acidity, with the Y7 strain dropping the pH the most significantly, reaching 3.42.
After 24 h of culture for each strain, the order of pH from largest to smallest was Y11>Y6>Y1> Y13> Y4>Y7 (Figure 3a).
After 24 hours of incubation, the results showed (Figure 3b): 6 organophosphorus-dissolving strains had some differences in their ability to secrete alkaline phosphatase.
The Y11 strain secreted alkaline phosphatase the strongest, reaching 38.69 μg mL-1h-1, and the second strain was the Y7 strain, reaching 36.20 μg mL-1h-1. According to the order of alkaline phosphatase activity from largest to smallest, Y11>Y7>Y4>Y13>Y6>Y1.
Their growth process can be divided into three stages: exponential growth, steady growth and gradual decline.
In the first 8 hours, bacteria grow exponentially rapidly due to colony growth and nutrient abundance, with a stable rate of 8-26 hours.
Peaking after 26 h, the growth rate gradually decreases due to nutrient limitation. Y1 and Y4 grow more slowly, while Y6, Y7, Y11, and Y13 grow at roughly the same rate (Figure 4).
The amplicon of 16SrDNA was sequenced (see Figure 5), with six strains of Acinetobacter, Pseudomonas, Enterobacteriaceae, and Panciforma species.
Y6 and Y13 are enterobacteria, and there is a strong connection between the two; Y11 and Y4 have great phylogenetic similarities, they belong to a taxon.
Phosphorus-dissolving bacteria are the key drivers of soil phosphorus conversion, and their distribution in soil has rhizosphere effect, and is closely related to soil type, environmental factors, plant species and other factors.
We screened 15 degradable organophosphorus bacteria from the rhizosphere soil of OsPT4, of which 6 were further screened and purified, and were able to grow stably in a higher concentration of organophosphorus and produce significant "transparent rings".
Preliminary work showed that the bacteria with phosphorus-dissolving function obtained by preliminary screening would lose the phosphorus-dissolving function in the later isolation, which was related to the growth adaptation of bacteria and the demand for phosphorus.
In addition, the growth characteristics and viability of the six bacteria did not change much, and their growth curves were consistent with the microbial culture characteristics.
In various environments, the solubility of phosphate by each bacterium is different, and the solubility of phosphate by each bacterium is also inconsistent.
At present, the dissociation mechanism of phosphorus by phosphorus-dissolving bacteria has been confirmed to be two: one is that some bacteria can reduce the pH of their environment by releasing acids such as organic acids, sulfides and H+, and then decompose them into phosphate that is insoluble in water.
Second, some bacteria under phosphorus stress will produce a lytic enzyme that can break down organophosphorus extracellular phosphate.
The detection of pH and ALP activity of bacterial liquid after 24h incubation showed that the phosphorus lysis mechanism of Y7 strain was mainly acidification and ALP.
The main mechanism of phosphorus lysis in Y11 strain is ALP action, while there is no obvious difference in the secretion of ALP activity in other strains, and there is a certain correlation between pH decrease and phosphorus dissolving ability.
Phosphorus-dissolving bacteria prevalent in farmland soil include: Bacillus, Acinetobacter, Pseudomonas, Escherichia, Irwinella, Serratia, Enterobacteriacetes, etc.
In the early stage, our research group isolated 6 bacteria that can effectively degrade organophosphorus from OsPT4 rhizosphere soil, among which Y1 is Acinetobacter, Y6 and Y13 are Enterobacteriaceae, Y4 and Y11 are Pseudomonaspyloris, and Y1 is Pacnes.
Although a new type of phosphorus-dissolving fungus (Y7) isolated from paddy soil has not been reported, it is known to be ubiquitous in nature and used in the cultivation of rice, barley, radish and other fields.
epilogue
In this study, 15 bacteria that can decompose organophosphorus were screened from the rhizosphere soil layer of rice containing phosphorus efficiency genes, and 6 bacteria that could decompose clear rings were obtained.
The study found that the phosphorus-dissolving performance of six strains was similar to that of several known phosphorus-dissolving bacteria in the field, represented by Y7, with the highest solubility, and 87.43mgL-1 could be decomposed in 24 hours, which was a new strain with strong phosphorus-dissolving potential.
Among them, Y7 is the most commonly used plant endophytic fungus in rice grain, and its phosphorus lysis mechanism is mainly based on the decomposition of acidified bases.
In addition, whether the application of phosphorus-efficient genotype rice can regulate the population composition of phosphorus-dissolving bacteria in the soil, and then increase the population size of the soil, can be further explored by modern molecular biology.
Bibliography:
[1] Liu Zhiyu, "Phosphorus nutrition of plants and bioavailability of soil phosphorus"
[2] Li Shiqi, Li Zheng, Wang Xianning, et al. Research Progress on the Physiological and Ecological Process of Nitrogen and Phosphorus Absorption and Utilization by Plants
[3] Wang Qingren, Li Ji, Li Zhensheng, "Plant nutrition research on efficient use of soil phosphorus"
[4] Wang Tao, Yang Yuanhe, Ma Wenhong, "Size, distribution and influencing factors of soil phosphorus reservoirs in China"