Effective separation method of Ustilaginoidea virens (Cooke) Takahashi capable of producing five ustiloxins

Abstract

The invention relates to a separation method of Ustilaginoidea virens (Cooke) Takahashi, and especially an effective separation method of Ustilaginoidea virens (Cooke) Takahashi capable of producing five ustiloxins. The effective separation method comprises a step of false smut ball collection and a step of strain isolation. According to the effective separation method, collected false smut balls are immersed in 75% alcohol for 10s, and are subjected to spontaneous combustion for 5s using an alcohol lamp; water on the surface is removed using sterilized filter paper; surface tissue is cleared under sterile operation conditions; the false smut balls are cut using a sterile scalpel; middle layers are collected, and inoculation on sterile PSA plating medium containing 0.05g / L chloromycetin is performed; after inoculation, obtained culture dishes are subjected to incubation at a constant temperature of 28 DEG C in an incubator; and bacterial strain preservation is carried out after strain purification. Isolation success rate of the effective separation method on collected fresh single yellow, yellow green, or green false smut balls is about 100%; isolation success rate on single yellow, yellow green, or green false smut balls stored at 4 DEG C for 4 months or shorter is 90% or higher, and isolation success rate on single yellow, yellow green, or green false smut balls stored at -70 DEG C for 1 month or shorter is 80% or higher.

Description

technical field [0001] The invention relates to a method for isolating Chlorococcus oryzae, in particular to an effective method for isolating Chlorococcus oryzae producing five kinds of rice koji toxins. Background technique [0002] Rice false smut is a fungal disease of rice, which is caused by the infection of the panicle of rice by Ustilaginodea virens (Cooke) Takahashi. Rice false smut is relatively common and occurs in many countries such as China, Japan, the United States, India, and the Philippines, and is a worldwide rice disease (Fu et al, 2015; Ashizawa et al, 2010; Hamed et al, 2014). Before the 1980s, rice false smut was only sporadically distributed in middle and late rice fields, and it was a secondary disease of rice; however, with changes in rice varieties, farming methods, fertilizer dosage, and climate and environment, in recent years, rice false smut has changed from the past The sporadic distribution of rice has transformed into one of the major diseas...

AI Technical Summary

Problems solved by technology

In the chlamydospore suspension method, rice koji balls can be prepared into chlamydospore suspensions after being washed with sterile water. Since the surface layer of rice koji balls is accompanied by a large number of other pathogenic bacteria, their growth rate is faster than that of rice koji bacteria. It is easy to contaminate rice smut bacteria, and the miscellaneous bacteria that grow too fast cover a small amount of steamed bun-shaped rice smut bacteria colonies, which increases the difficulty of separation and purification

The disadvantage of the rice ball method is that a variety of pathogenic bacteria are associated with the rice ball, and they can also grow rapidly during the cultivation process and contaminate the target bacteria.

The sclerotia germination method is relatively easy to succeed. Studies have proved that sclerotia is the first choice for the efficient isolation of rice smut. However, the formation of sclerotia is limited by environmental conditions, and it is difficult to collect and obtain sclerotia. Moreover, it is rare in southern rice areas. This method is used to isolate southern rice Aspergillus is not effective

Spore shake-off method, because the surface of rice koji is not disinfected, a large number of bacteria on the surface of rice koji are easy to grow rapidly on the surface of the medium, which affects the germination, growth and purification of chlamydospores, and is as susceptible as the chlamydospore suspension method. Contaminated by other pathogens, it is not easy to successfully obtain strains of rice false smut

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