	US 12,185,674 B2
	Method for inhibiting microbial clogging in drip irrigators
Yunkai Li, Beijing (CN); Peng Song, Beijing (CN); and Hongxu Zhou, Beijing (CN)
Assigned to CHINA AGRICULTURE UNIVERSITY, (CN)
Appl. No. 17/598,747
Filed by CHINA AGRICULTURE UNIVERSITY, Beijing (CN)
PCT Filed Mar. 29, 2019, PCT No. PCT/CN2019/080390 § 371(c)(1), (2) Date Sep. 27, 2021, PCT Pub. No. WO2020/198926, PCT Pub. Date Oct. 8, 2020.
Prior Publication US 2022/0322620 A1, Oct. 13, 2022
Int. Cl. A01G 25/02 (2006.01); C12N 1/20 (2006.01); C12Q 1/10 (2006.01)

CPC A01G 25/02 (2013.01) [C12N 1/205 (2021.05); C12Q 1/10 (2013.01)]

7 Claims

1. A method for inhibiting microbial clogging of a drip irrigation emitter when the clogging degree of the drip irrigation emitter reaches at least 50%, comprising the following steps:

i) determining a key bacteria causing the clogging of the drip irrigation emitter using developmental molecular ecological network analysis (MENA), via a MENA network analysis system capable of calculating a correlation among an array of bacteria, to construct and analyze a phylogenetic molecular ecological network;

ii) performing screening of an antagonistic bacteria to inhibit the clogging of the drip irrigation emitter;

wherein the steps of using developmental molecular ecological network analysis to determine the key bacteria causing the clogging of the drip irrigation emitter and the screening of an antagonistic bacteria comprise:

a. cultivating an epiphytic biofilm located in a channel of the drip irrigation emitter,

b. extracting a sample of microorganisms in the epiphytic biofilm,

c. extracting the total DNA of the sample of microorganisms in the epiphytic biofilm,

d. expanding and purifying the V3-V4 variable region of 16S rRNA originating from the total extracted DNA of step ii) c.,

e. quantitatively detecting and sequencing the V3-V4 variable region of 16S rRNA to obtain an RNA sequence listing,

f. applying the sequencing result of the sample of microorganisms to construct a phylogenetic molecular ecological network,

g. determining the key bacteria causing the clogging based on the correlation among the array of bacteria; at the same time, according to the crops to be irrigated, selecting a candidate bacteria having biological control function from the array of bacteria, and

h. combining the key bacteria and the candidate bacteria to conduct an antagonism experiment; wherein the strains in the candidate bacteria having an antagonistic effect against the key bacteria are used as a selected antagonistic bacteria;

iii) fermenting the selected antagonistic bacteria in a medium containing a fresh biogas slurry, wherein the steps to generate the selected antagonistic bacteria include:

a. concentrating the fresh biogas slurry by spacing the fresh biogas slurry into a reactor in a vacuum state for concentration using a concentrating pressure of 1×10⁵Pa, wherein the fresh biogas slurry is concentrated to a moisture content of about 60%, and

b. fermenting and cultivating the fresh biogas slurry and a pathogenic microorganism to generate the selected antagonistic bacteria; and

iv) applying the selected antagonistic bacteria to a drip irrigation system, wherein the application is a periodic application with a frequency of 1 time every 1 to 2 weeks, and 2 to 3 hours of each application.