NL2031981B1

NL2031981B1 - Small-hole isolation method for rapid removing bacterial contamination in fungus

Info

Publication number: NL2031981B1
Application number: NL2031981A
Authority: NL
Inventors: Qiu Haiping; Sun Guochang; Zhang Zhen; Chai Rongyao; Mao Xueqin; Shi Xiaoxiao; Wang Yanli; Wang Jiaoyu
Original assignee: Zhejiang Acad Agricultural Sci
Priority date: 2022-05-25
Filing date: 2022-05-25
Publication date: 2023-06-28

Abstract

Small-hole isolation method for rapid removing bacterial contamination in fungus The present invention discloses a small-hole isolation method for quickly removing bacterial contamination in a fungus, including the following steps: a. preparing a fungal culture medium, sterilizing the fungal culture medium at 121°C for 15 minutes under a high temperature and a high pressure, pouring the fungal culture medium into a sterile petri dish, waiting for the culture medium to be solidified, and cutting one or more square or circular small holes with a diameter of about 3-5 mm on a culture medium plate; b. picking a sample with a toothpick into the hole cut in Step a, and a size of the sample being less than the square or circular small hole in Step a; c. covering the culture medium with a sterile cover glass slip, gently pressing around the cover glass slip to be in close contact with the culture medium, so that no air bubbles remain between the culture medium and the cover glass slip; d. placing the culture medium treated in Step c in a constant temperature and constant humidity incubator for 7-10 days; e. picking up a newly grown fungal mycelium around the cover glass slip with the toothpick, and re-inoculating the fungus on a new fungal culture medium for cultivation; and f. observing a fungal colony that the new fungal culture medium in Step e further cultivates and grows. After 2-3 cyclic operations according to the forgoing method, complete purification can be achieved. The method is simple in operation and small in workload, and is suitably used for various bacterial contamination. A comparative experiment shows that an effect of the method is obviously better than that of suppression and sterilization of antibiotics.

Description

Specification

SMALL-HOLE ISOLATION METHOD FOR RAPID REMOVING BACTERIAL CONTAMINATION IN

FUNGUS

Technical Field

The present invention relates to the field of microorganisms, in particular to removal of bacterial contamination in fungus under a condition of a solid culture medium plate.

Background Art

In a process of fungal culture, contaminative microbes are everywhere. Common microorganisms that cause contamination include bacteria, fungi, mycoplasmas, and mites. A fungus such as mold and yeast grow rapidly under a culture condition. Contamination that the fungus cause can be easily observed. Bacterial species can also be identified based on colony characteristics and microscopic observation methods of the fungus on the medium, while bacterial contamination has a certain incubation period and is manifested even after several generations of culture. Therefore, the removal of bacteria is more difficult and important.

Bacterial contamination is mostly a mucous plaque, and mainly caused by Bacillus,

Escherichia coli and other bacteria. According to a location of bacteria, the bacteria can be divided into surface bacterial contamination and endogenous bacterial contamination. Surface bacterial contamination is often manifested in 1-2 days after inoculation, while contamination caused by endogenous bacteria is not obvious at the time of inoculation. With an increase in the number of culture transfers, a bacterial amount gradually accumulates and only appears on the culture medium. At present, a method for eliminating bacterial contamination is mainly to use antibiotics on the culture medium, Meanwhile, separation and contamination removal according to specific fungal species and a growth rate of bacterial contamination have certain effects. In addition, it has also been reported that the presence of an organic matter in the culture medium is an important cause of the contamination, so removing the organic matter in the culture medium is a way to reduce the contamination. Although a plurality of methods by using such as the antibiotics and reduction of the organic matter can currently reduce the contamination, these treatment methods have more or less respective shortcomings for the removal of a plurality of bacteria.

The antibiotics are often added to the culture medium to prevent the contamination, or to inhibit bacterial growth or destroy the bacteria if the bacterial contamination is identified.

However, due to diversity of bacterial and fungal species, a type and a concentration of the antibiotics used are changed dramatically. In addition, the antibiotics have different bacteriostatic spectrums. There is no antibiotics that are effective against all bacteria, and an efficacy period of the antibiotics is short. The antibiotics are generally unstable. In case of acid, alkali or heating, the antibiotics are easy to decompose and lose activity. A single antibiotic is bacteriostatic, and is easy to produce drug resistance. Once the antibiotics are stopped to use, a contamination rate increases significantly. A high concentration of the antibiotics affects the growth of plants. In view of this, antibiotics can only be used as an auxiliary measure to prevent contamination. Of course, people add double antibiotics for a better bacteriostatic effect, that is, two antibiotics against Gram-positive bacteria and against Gram-negative bacteria, but the bacteriostatic effect of the double antibiotics sometimes do not have an obvious effect. In conclusion, the inhibition of bacteria by the antibiotics lacks stability and efficiency. A method for reducing the organic matter in the culture medium to inhibit growth of the bacteria also affects the fungus or a desired strain to a certain extent, so the method is not preferred.

Summary of the Invention

For lack of an efficient and stable bacteria removal system, the objective of the present invention is to provide a new method for rapidly removing bacteria, especially removing bacterial contamination in a fungus under a condition of a solid culture medium plate, which is greatly restricted by an antibiotic, and whose bacteriostatic effect is not obvious.

The present invention provides a small-hole isolation method {a CS method) for rapidly removing bacterial contamination in a fungus, comprising the following steps: a. preparing a fungal culture medium, sterilizing the fungal culture medium at 121°C for 15 minutes under a high temperature and a high pressure, pouring the fungal culture medium into a sterile petri dish, waiting for the culture medium to be solidified, and cutting one or more square or circular small holes with a diameter of about 3-5 mm on a culture medium plate; b. picking a sample with a toothpick into the small hole cut in Step a, and a size of the sample being less than the square or circular small hole in Step a; c. covering the culture medium with a sterile cover glass slip, gently pressing around the cover glass slip to be in close contact with the culture medium, so that no air bubbles remain between the culture medium and the cover glass slip; d. placing the culture medium treated in Step c in a constant temperature and constant humidity incubator for 7-10 days; e. picking up a newly grown fungal mycelium around the cover glass slip with the toothpick, and re-inoculating the fungus on a new fungal culture medium for cultivation; and f. observing a fungal colony that the new fungal culture medium in Step e further cultivates and grows.

The beneficial effects of the present invention are as follows: First, the present invention can reduce or even avoid use of an antibiotic, get rid of cognition brought by a traditional method, and avoid generation of bacterial resistance. Second, experimental materials involved in the present invention are very simple and can be easily obtained basically. A whole experimental process is simple to operate, with a high success rate. The method has a relatively short period and a small workload.

Detailed Description of Embodiments

Embodiment 1

Materials to be tested:

Magnaporthe oryzae pathogen: A strain of Magnaporthe oryzae was a strain of

Magnaporthe oryzae Guy-11, which was preserved by the fungal disease room of the Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences. Magnaporthe oryzae Guy-11 in the embodiment was contaminated with Escherichia coli (Escherichia coli, E. coli for short).

An experiment was performed based on the following steps: {1} Preparation of a culture medium: According to a growth rate of Magnaporthe oryzae in a culture medium suitable for fungal cultivation, this method was screened to be used for a CM culture medium for removing bacterial contamination in a fungus. The culture medium (CM) of a plate culture: 10 g of glucose, 2 g of peptone, 1 g of yeast extract, 1 g of casamino acid, 50 mL of nitrogen salts (20xNitrate salts; trace elements; vitamins), and 18 g of agar. ddH20 had 1000 mL of a constant volume. pH of 1 M NaOH solution was adjusted to 6.5. Sterilization was performed by autoclaving at 121°C for 15 minutes. (2) Pre-treatment of the culture medium: one square or circular small hole with a diameter of about 3-5mm from the medium plate obtained in Step (1) with a toothpick or a hole punch, and a sterilized cover glass slip was prepared for stand-by use. (3) Inoculation: An appropriate amount of Magnaporthe oryzae Guy-11 mixed with

Escherichia coli was carefully picked into the small hole cut in Step (2) with the toothpick. (4) Covering the culture medium with the cover glass slip: After inoculation, the sterilized cover glass slip was picked up with sterile tweezers and the culture medium was covered with the cover glass slip carefully, a periphery of the cover glass slip was gently pressed to be in close contact with the culture medium, so that no air bubbles remain between the culture medium and the cover glass slip; (5) Cultivation: The culture medium treated in step {4} was cultured at 28°C for 7 days. (6) Checking growth of the fungus. After a mycelium grows around the cover glass slip, a newly grown fungal mycelium was inoculated on a new CM culture medium for cultivation with a sterile toothpick. A new formed colony was Magnaporthe oryzae Guy-11 strain with the bacterial contamination removed.

T1: CS treatment without antibiotics (Antibiotic-free with holes}; T2: CS treatment on the culture medium with the antibiotics {Antibiotics with holes); T3: Bacterial suppression with antibiotics alone {Antibiotics without holes); T4: Subculture on the culture medium containing no antibiotics (Antibiotic-free without holes).

The present invention can complete the removal of bacteria in about 7-10 days. The mycelium of the Magnaporthe oryzae Guy-11 inoculated in the small hole grew out and gradually expanded on an edge of the cover glass slip through the small hole, while the mixed

Escherichia coli could not pass through the culture medium and remained in an enclosed space formed by the cover glass slip and the small hole. The newly grown mycelium of the

Magnaporthe oryzae Guy-11 around the cover glass slip was re-inoculated on a new CM culture medium with the toothpick for cultivation. Traits of a colony grown in this way were significantly better than those of the initial strains, which played a very good purification effect. No difference was provided in an effect between T1 and T2. After treatment, the Magnaporthe oryzae Guy-11 strain grew well on the culture medium, the colony was plump, the color was uniform, and sporulation and other traits were restored, and the colony was well purified. At the same time, T1 saves the use of the antibiotics relative to T2. After T3 treatment was suppressed with the antibiotics, the bacterial contamination was improved, but the colony could not be completely purified and the traits could not be completely restored. The T4 treatment was a simple subculture on a culture medium without the antibiotics. The bacterial contamination of the Magnaporthe oryzae Guy-11 strain could not be improved. A growth rate and morphology of the colony were limited, and the Magnaporthe oryzae Guy-11 strain could no longer be used for scientific research and teaching. Genomic DNA was extracted from the

Magnaporthe oryzae Guy-11 strain obtained after T1, T2, T3, and T4 treatments, and PCR detection was performed using an Escherichia coli-specific primer. Results showed that no 5 fragments of Escherichia coli DNA could be detected in the fungus after T1 and T2 treatments.

PCR detection results further verified that a small-hole isolation method (a CS method) of the present invention could effectively remove Escherichia coli in the Magnaporthe oryzae Guy-11 strain.

Embodiments 2, 3, 4, and 5

An Magnaporthe oryzae Guy-11 strain, a Magnaporthe oryzae 2539 strain, a Magnaporthe oryzae 70-15 strain, and a Magnaporthe oryzae TH3 strain that were contaminated by

Agrobacterium tumefaciens were selected as objects, and the others were the same as in

Example 1. The Magnaporthe oryzae Guy-11 strain, the Magnaporthe oryzae 2539 strain, the

Magnaporthe oryzae 70-15 strain, and the Magnaporthe oryzae TH3 strain were subjected to a removal experiment of Agrobacterium tumefaciens. No difference was provided in an effect between T1 and T2. The Magnaporthe oryzae Guy-11 strain, the Magnaporthe oryzae 2539 strain, the Magnaporthe oryzae 70-15 strain, and the Magnaporthe oryzae TH3 strain were purified after treatment, respectively. A growth rate of the strains on the culture medium, colony status, sporulation and other traits were restored and improved. Compared with T2, T1 saved the use of the antibiotics. After T3 treatment, a partial improvement was provided, but a fungal colony could not be completely purified. After T4 treatment, the bacterial contamination and culture properties of the Magnaporthe oryzae Guy-11 strain, the Magnaporthe oryzae 2539 strain, the Magnaporthe oryzae 70-15 strain, and the Magnaporthe oryzae TH3 strain were not improved, and the Magnaporthe oryzae Guy-11 strain, the Magnaporthe oryzae 2539 strain, the

Magnaporthe oryzae 70-15 strain, and the Magnaporthe oryzae TH3 strain could no longer be used for scientific research and teaching. Genomic DNA was extracted from the strains of T1,

T2, T3 and T4, and PCR detection was performed using an Agrobacterium tumefaciens-specific primer. Results showed that fragments of Agrobacterium tumefaciens DNA could not be detected in fungus after T1 and T2 treatments. The PCR detection results further verified that a small-hole isolation method (CS method) of the present invention could effectively remove

Agrobacterium tumefaciens in the Magnaporthe oryzae Guy-11 strain, the Magnaporthe oryzae

2539 strain, the Magnaporthe oryzae 70-15 strain, and the Magnaporthe oryzae TH3 strain.

Embodiments 6 and 7:

Fusarium graminearum and Colletotrichum gloeosporioides contaminated by bacteria were selected as objects, respectively, with PDA as a substratum, the others were the same as

Embodiment 1, respectively. Removal experiments of Agrobacterium tumefaciens were performed for mold bacteria and Colletotrichum gloeosporioides bacteria. No difference was provided in an effect between T1 and T2. The treated Fusarium graminearum and

Colletotrichum gloeosporioides grew well on the culture medium, respectively. Colonies were purified, and sporulation and other traits were restored. After T3 treatment, the strain was partially improved, but a fungal colony could not be completely purified. After T4 treatment, the bacterial contamination and culture properties of Fusarium graminearum and

Colletotrichum gloeosporioides were not improved, and Fusarium graminearum and

Colletotrichum gloeosporioides could no longer be used for scientific research and teaching.

Embodiments 8 and 9:

Rhizoctonia solani and Botryosphaeria rhodina contaminated by bacteria were taken as objects respectively. MEA was taken as a culture medium, and the others are the same as

Embodiment 1, respectively. Removal experiments of Agrobacterium tumefaciens were performed for Rhizoctonia solani and Botryosphaeria rhodina, respectively. No difference was provided in an effect between T1 and T2. After treatment, Rhizoctonia solani and

Botryosphaeria rhodin grew well on the culture medium. Colonies were purified, and sporulation and other traits were restored. After T3 treatment, the strain was partially improved, but a fungal colony could not be completely purified. After T4 treatment, the bacterial contamination and culture properties of Rhizoctonia solani and Botryosphaeria rhodina were not improved, and Rhizoctonia solani and Botryosphaeria rhodina could no longer be used for scientific research and teaching.

For all fungi tested, a sterilization effect of a small-hole isolation method (a CS method) was far better than that of antibiotics. A whole experiment was simple, with a short cycle and a good effect, andwithout easiness to repeat, and costs were saved without the antibiotic.

Moreover, in the present invention, the bacteria were artificially added by the present inventor.

In practice, the bacterial content of bacterial contamination was less than this added amount in most cases. Therefore, in practical application, a removal effect of the bacteria was very ideal. In application, a method of the present invention was used to remove the bacteria for at least 300 times, including different fungal species and different bacterial contamination. Bacteria mixed in fungal colonies that were difficult to distinguish and bacteria that were difficult to remove by an ordinary antibiotic method were provided Most of the bacteria contained in these contaminated fungi could be removed by the CS method one time, and respective bacteria need to be treated twice or three times to achieve an ideal sterilization effect. Respective culture traits of the fungal strains after bacteria were removed could be restored to normal, even better than the original strains without contamination.

At present, a laboratory subculture of a plant pathogenic fungus, including Magnaporthe oryzae, had common problems such as strain degradation, reduced sporulation, and weakened pathogenicity. The reasons for these problems were not clear, and may be related to gene expression, post-translational modification and other processes. Therefore, in research, it was necessary to preserve original strains for a long time, try to having the subculture as little as possible, and rejuvenate the strains regularly. In the past, the rejuvenation of the strains was generally re-inoculated on a corresponding host, and a whole process was complicated and a cycle was long. The present invention found that after the CS treatment, in addition to the removal of contamination, other traits of the fungus, such as sporulation, melanin, pathogenicity, etc., were improved, while antibiotic treatment seldom produced such effects. A reason for this might be that in a small hole of the culture medium, the fungus was in an environment containing bacteria on the one hand, and on the other hand had to break free from shackles of a cover glass slip. In such a case, the fungus that could successfully grow the cover glass slip had good traits. In short, the whole process is to remove the bacteria while allowing the fungus to simply rejuvenate, and the CS method is much simpler than the strains that were tied back and then isolated on a host.

Different bacteria had different abilities to grow and move on and within the culture medium due to differences in breathing patterns and motility. In theory, anaerobic bacteria and bacteria with flagella were able to grow and expand inside a solid culture medium. For the two bacteria used in this experiment, a metabolic type of Escherichia coli was heterotrophic facultative anaerobic type, and Agrobacterium tumefaciens was an aerobic type. Both of the two bacteria had flagella. Both of the two bacteria were well removed in different fungal strains by the CS method, indicating that motility of the bacteria generally did not significantly affect an effect of the CS method. In the application, a newly grown mycelium around the cover glass slip was transferred in time, and a tip of the mycelium was cut as much as possible. In a word, the

CS method disclosed in the present invention was a bacterial removal method with easy operation, a good effect and wide application.

Claims

CONCLUSION

1. Small hole isolation method to quickly remove the bacterial contamination from mold, containing the following steps:

a. Prepare a fungal culture medium, sterilize the fungal culture medium at 121°C for 15 minutes under high temperature and high pressure, pour the fungal culture medium into a sterile petri dish, wait for the culture medium to solidify, and cut one or more small square or round holes with a diameter of about 3-5 mm on a medium plate;

b. picking up a sample with a toothpick in the small holes cut in step a, where a size of the sample is smaller than the square or round small hole in step a;

c. covering the culture medium with a sterile cover slip, gently pressing around the cover slip to bring it into close contact with the culture medium so that no air bubbles remain between the culture medium and the cover slip;

d. placing the culture medium treated in step c in a constant temperature, constant humidity incubator for 7 to 10 days;

e. pick up with the toothpick a newly grown mycelial fungus around the coverslip lip, and re-inoculate the fungus on a new fungal culture medium for cultivation; and f. observing a fungal colony further cultivated and growing in the new fungal culture medium in step e.

The small-hole isolation method for quickly removing the bacterial contamination from molds according to claim 1, wherein the one or more small square or round holes with a diameter of about 3-5 mm are cut out by using a sterile toothpick or a perforator .

The small hole isolation method for quickly removing the bacterial contamination from molds according to claim 1, wherein the bacteria include Escherichia coli or agrobacterium.

The small hole isolation method for quickly removing the bacterial contamination from molds according to claim 1, wherein the molds contain Magnaporthe oryzae, gibberella saubinetii, Colletotrichum gloeosporioides, riziocotinia solani or botryosphaeria dothidea.