Population Dynamics Of Bacillus Subtilis NCD-2 In Cotton Soil Under Drip Irrigation

Cotton verticillium wilt（CVW）is a serious soil-borne disease caused by Verticillium dahliae Kleb.At present,the integrated management of CVW based on biological control have attracted more and more attention and played an extremely important role in the prevention and control of CVW.In the biological control of CVW,microbial fungicides is the important biological control agents.Bacillus subtilis NCD-2 is an antagonistic strain isolated from cotton rhizosphere soil,showing promising control effect on CVW.The microbial fungicide based on this strain as the main ingredient has registered officially in China in 2010（PD20101654）.This experiment simulates the drip irrigation technology in Xinjiang cotton fields to investigate the colonization dynamics of strain NCD-2 in soil,cotton rhizosphere and root surface soils under drip irrigation conditions,and the control effect on CVW.（1）The marker strain NCD-2^Rifif which could normally grow on 300μg/mL rifampicin medium was selected by gradient induction.The colony morphology and growth curve of this strain showed no significant difference from the wild strain.（2）The number of marker strain NCD-2^Rif in different deep layers was detected by using a resistant plate colony counting method.The results showed that when the strain NCD-2^Rifif was dripped into the soil by drip irrigation,the bacterial content varied with drip irrigation time and soil depth.On the 21st day after drip irrigation,the number of bacterial cells reached the peak at 5 cm and 10 cm,and then the number of bacterial cells dropped sharply.The number of bacterial cells in 5 cm soil layer tends to be stable after the 35th d of drip irrigation,and which in 10 cm soil layer tends to be stable after the 28th d of drip irrigation.After the 14th d of drip irrigation,the bacteria amount reached its peak at 15 cm and 20 cm,and then the number of bacteria decreased sharply.the number of bacteria in 15cm soil layer tended to be stable after the 21st d of drip irrigation,and which in 20 cm soil layer tends be stable after the 35th d of drip irrigation.On the first day after drip irrigation,the number of bacteria at 25 cm and 30 cm peaked,and the number of bacterial cells dropped sharply.The number of bacterial cells in 25 cm soil layer tends to be stable after the 35th d of drip irrigation,and which in 30 cm tends to stabilize after the 28th d of drip irrigation.It can be seen that with the increase of soil depth,the bacterial number of the marker strain NCD-2^Rif in soil showed a decreasing trend,with the extension of sampling time,the number of bacterial strains of the marker strain NCD-2^Rifif in each layer of soil also showed a downward trend.（3）The bacterial strain NCD-2^Rif was inoculated by drip irrigation with antibiotic resistance marker combined with pot test,and the colonization number of the marker strain NCD-2^Rif in cotton rhizosphere soil and root surface was measured at different time intervals after inoculation.The results showed that the marker strain NCD-2^Rif was able to colonize both the rhizosphere soil and the root surface of cotton,and the number of bacteria in which reached the peak on the 1st day after inoculation.After the 35th day of inoculation,the colonization amount of the marker strain NCD-2^Rif in the rhizosphere soil and root surface can still reach a high level.At the same time,the bacterial number of the marker strain NCD-2^Rif in the cotton rhizosphere soil was higher than that in the root surface.With the prolongation of time,the bacterial count of the marker strain NCD-2^Rifif in cotton rhizosphere soil dropped sharply,while which on the root surface declined slowly.（4）Pot experiment was used to detect the control effect of Bacillus subtilis NCD-2WP on CVW under drip irrigation.On the basis of clarifying its safe use dose,the effect of different drip doses on the control of CVW was examined.After applying cotton seed with drip irrigation with 5 kg/m²,3 kg/m² and 1 kg/m² different doses of biocontrol agent,the emergence rates were 84.38%,80.73%and 79.17%respectively,and there was no significant difference in the emergence rate among the three treatments.Compared with the blank control,the emergence rate of the 5 kg/m² and 3 kg/m² treated cotton increased significantly,and the emergence rate of the 1 kg/m² treated cotton had no significant difference,indicating that the dose used in the preparation is safe to cotton.The control effect test results showed that 5 kg/m² bio-control agent was the highest,which was40.19%,the control effect of 3 kg/m² was 23.95%,and the control effect of 1 kg/m² was15.50%.It shows that with the increase of the dose of biocontrol agent,its control effect on CVW appears an increasing trend.