Screening and Taxonomic Status of a Highly Efficient Antifungal Strain against Cytospora chrysosperma

Chongwei LI , Fuqiang SONG, Ruiqing SONG

1. College of Forestry, Northeast Forestry University, Harbin 150040, China;

2. Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150080, China

In recent years, the occurrence of popular bark-rot disease caused by Cytospora chrysosperma Fr.has been increased continuously. In hardest-hit areas, the death of large pieces of forests often occurs, seriously affecting the development of forest industry and the natural barrier function of Three-North Shelterbelt[1-2].Traditional chemical pesticides suppress pathogens, as well as killing beneficial microorganisms in trees, affecting the biological diversity in forests. The long-term use of pesticides also causes drug resistance in pathogens.The exploration of efficient and safe biological control methods are still difficult issues for domestic and foreign experts. The studies on popular bark-rot disease are started many years ago in China.According to the literature,low temperature induced the occurrence of popular bark-rot disease in seedling beds in Jimsar County, Xinjiang in 1975[3]. Hu et al.[4]had effectively curbed the spread of popular bark rot disease by chemical methods. Wang et al.[5]deeply studied the symptoms and pathogen of popular bark-rot disease,which provided an important theoretical basis for later study.Xiang and Gao et al.[6]screened microorganisms which had antagonistic effect against Cytospora chrysosperma,and they tried to adopt biological means to control popular bark-rot disease. Gao et al.[7]carried out confrontation culture between T. harzianum T88, T. atroviride T95 and Cytospora chrysosperma, Dothiorella gregaria on plates and slides to investigate the effects of Trichoderma metabolites on pathogen colony and mycelium dry weights.Sun et al.[8]confirmed the presence of antibiotics in solid fermentation broth of Trichoderma SMF2,and they found that the antibiotics had good controlling effect onPseudomonas solanacearum.Ji et al.[9]isolated,purified and identified the antibacterial active ingredients from the broth of Lactarius vellereus, which provided a theoretical basis for future development of biological pesticides.

From the 29 domestic and foreign Trichoderma strains, this study screened the strains which showed highly efficient controlling effect on Cytospora chrysosperma Fr. The morphological and physiological characteristics and taxonomic statuses of the screened strains were studied so as to lay a theoretical foundation for the future isolation and identification of antibacterial active ingredients from the strains and the development of microbial pesticides.

Materials and Methods

Materials

Trichoderma strains A total of 29 Trichoderma strains were used in this study. Among them, 7 stains were introduced from foreign countries, and the others were all domestic. The trains were provided by the pathology laboratory of College of Forestry,Northeast Forestry University.

Pathogen strain The Cytospora chrysosperma Fr.strain was also preserved by the pathology laboratory of College of Forestry, Northeast Forestry University.

Media The used media included PDA medium and PD medium.

Methods

Screening of highly efficient Trichoderma strains Combing the antagonistic effect of Trichoderma strains on Cytospora chrysosperma Fr. and antibacterial activity of Trichoderma strains broth against Cytospora chrysosperma Fr., the screening of highly efficient antifungal strains were performed. For the antagonistic effect of Trichoderma strains on Cytospora chrysosperma Fr., confrontation culture was adopted[10]; for the antibacterial activity of Trichoderma strains broth against Cytospora chrysosperma Fr., growth rate method was adopted[10].

Screening of extracts from highly efficient Trichoderma strains Certain volumes (250 ml of each)of T-33 strain broth were extracted by different-polarity petroleum ether, chloroform, ethyl acetate, n-butanol, n-hexane and diethyl ether, respectively.The ratio between broth and organic solvent was 1∶3 (V/V) with extraction time of 48 h and extraction temperature of 25 ℃. After the extraction, the organic solvents were concentrated by reduced pressure in vacuo, and then the remaining extracts were diluted to one tenth of the original volume of culture filtrates with 10% Tween 80. The antibacterial activities of Trichoderma strains were determined by growth rate method and modified hanging drop method. There were three replicates for each treatment. The 10%Tween 80 was used as a control.The effects of extracts on mycelial growth of Trichoderma strains were detected using growth rate assay,and the effects of extracts on spore germination of Trichoderma strains were detected using modified hanging drop method[11].

Morphological and physiological characteristics of highly efficient antifungal strains The morphological and physiological characteristics of the Trichoderma strains were studied with the method described by Ji et al.

Taxonomic status of highly efficient antifungal strains The DNA in mycelia of T-33 strain was extracted with CTAB method, and the rDNA ITS sequences were amplified using universal primers ITS 1 (5’-TCCGTAGGTGAACCTGCGG-3’)and ITS 4(5’-TCCTCCGCTTATTGATATGC-3’).The universal primers were synthesized by the TaKaRa Biotechnology (Dalian)Co., Ltd. The PCR products were examined by 1.0% agarose (containing 0.5 μg/ml of EB) electrophoresis. The electrophoresis results were analyzed using the UV gel imaging system[12]. If clear strips of PCR products were shown on the electrophoregram, the extraction of DNA from mycelia of the T-33 strain was proved to be successful. A certain amount of T-33 strain broth was sampled for the bio-directional sequencing using primers T7(5’-TCCgTggTgAACCTgCgg-3’) and SP6 (5’-TCCTCCgCTTATTgATATgC-3’) for the vector. The sequencing was completed by the Sangon Biotech(Shanghai) Co., Ltd. The used sequencing instrument and reagent were ABI PRISM3730 and BigDye Terminator v3.1, respectively. The obtained ITS sequence was submitted to Nucleotide BLAST and Translated BLAST Searches, respectively, and then the comparison between ITS sequence and obtained homologous sequences was performed. The length and GC nucleotide content in the obtained ITS sequence were analyzed by the Nucleotide Compisition of Sequence of BioEdit 5.06. From the GenBank database, the ITS sequences of high-similarity and highhomology species, especially the Trichoderma spp. were obtained, and then the phylogenetic relationships between T-33 and the homologous species were analyzed. The phylogenetic tree for the ITS region (ITS1+5.8S+ITS2)was constructed by UPGMA method.

Results and Analysis

Screening results of highly efficient Trichoderma strains

After a 60-h confrontation culture,the T-33(39.9%),T-14(21.5%)and T-09 (20.6%) all showed relatively high inhibition rates,and the T-33 (85.1%),T-14(83.1%)and T-15(69.6%)strains showed relatively high growth inhibition rates (Fig.1,Fig.2).The inhibiting effects of the organic solvent extracts of strain broths on mycelial growth and spore germination of pathogen were shown in Table 1. The n-butanol extract showed the highest inhibiting rate(94.2% ) on mycelial growth of pathogen, followed by the ethyl acetate extract (32.3%). The n-butanol extract of T-33 broth significantly damaged the mycelia of pathogen (Fig.3,Fig.4).Based on the above results,the T-33 strain was selected as highly efficient strain for further screening.

Morphological characteristics of highly efficient Trichoderma strains

T-33 strain grew rapidly on PDA plate, and it was radial. In the early stage,the colonies were white, dense,round and spread upwards and around until they covered the entire plate. In different seasons, the conidiation time was different. From April to June, yellow-green or green colonies were shown in the center of colony population 3-4 d after the inoculation, and finally, the entire colonies all turned green (Fig.5). From December toFebruary next year, the back of the colonies began to turn black one week after the inoculation, and none green colonies were observed on the front.So T-33 strain growth is affected by different season, and it does produce spores in winter.

Table 1 Effects of different extracts on mycelial growth and spore germination of pathogen

Under conditions of appropriate culture temperature and humidity,mycelia grew rapidly, and the colonies could be spread throughout the plate within a few days. The mycelia could grow in the temperature range of 4-42℃, and the spores could germinate in the temperature range of 10-30 ℃.Within the appropriate temperature range,high temperature was favorable for mycelial growth and spore germination. Spore germination requires a relative humidity higher than 50%,and spores germinated slowly in a dry environment.

After a 2-d liquid culture,the broth remained as a clear state, and sparse mycelia grew out. On the 3rdd since the inoculation, suspended solids appeared in the broth, and the broth seemed a little muddy. On the 4th d,mycelial balls appeared and became more and more, and when the broth was stood, the entire broth was filled with grain-sized mycelial balls, and only a thin layer of clear liquid was shown on the broth surface (Fig.6).In the spring, the broth turned green within 4-5 d,and in the other seasons,the broth was all pale yellow.

Physiological characteristics of highly efficient Trichoderma strains Effect of carbon source on growth of strains T-33 strain could grow on all the media with different carbon sources (Fig.7). It showed the best growth on the medium with glucose as carbon source, and the average colony diameters reached 2.81 and 7.41 cm after 24- and 48-h culture. So glucose was selected as the optimum carbon source.

Effect of nitrogen source on growth of strains T-33 strain could grow on all the media with different nitrogen sources (Fig.8). It grew best on the media with soy flour as the nitrogen source,and the average colony diameters were 3.5 and 8.6 cm after 24-and 48-h culture. Therefore, the soy flour was selected as the nitrogen source.

Effect of carbon source content on growth of strains The content of glucose, as the optimum carbon source, should be controlled with a range of 0.2%-3%. With the increase of glucose content, the strain growth was accelerated. When the glucose content reached 4%, the strain growth rate was decreased (Fig.9). When the glucose content was 3%, the average colony diameters were 3.62 and 8.21 cm, respectively after 24- and 48-h culture. So 3% (g/L) was selected asthe optimum content for glucose.

Effect of nitrogen source content on growth of strains The growth rates of T-33 strain were different on the PDS plate media with different contents of soy flour (Fig.10). When the soy flour content was 0.02%, the growth of the train was best, and after 24- and 48-h culture, the average colony diameters reached 4.22 and 8.93 cm. So 0.02% (g/L) of soy flour was selected as the optimum nitrogen source.

Effect of initial pH on growth of strains The strain could grow on all the PDA plate media with initial pH range of 4-9 (Fig.11). It showed the best growth on the PDA plate medium at pH 6,and the average colony diameters reached 3.45 and 8.54 cm, respectively after 24- and 48-h culture.Therefore, the optimum initial pH of medium for strain growth was 6.

Effect of culture temperature on growth of strains The strain could grow in the culture temperature range of 10-35 ℃ (Fig.2). The growth of mycelia was relatively good in the culture temperature range of 20-30 ℃.Especially,under the culture temperature of 25 ℃, the growth of mycelia was best and fastest. The average colony diameters were 3.96 and 8.89 cm, respectively after 4- and 5-d culture.Therefore,for T-33 strain,the optimum culture temperature was 25 ℃.

Taxonomic status of highly efficient strain T-33

The target fragment in the T-33 rDNA was amplified using primers ITS1 and ITS4, and its full length was 583 bp (Fig.13, Fig.14). The GenBank accession number of the target fragment was JF823649.

The obtained sequence was searched by Nucleotide BLAST and Translated BLAST Searches, and it was compared with the obtained homologous sequences. The cluster analysis results showed that there was certain genetic differentiation among Trichoderma spp. The similarity rate between T-33 strain and Trichoderma viride (accession number GU325689)was highest (99.9% ), so the T-33 strain was identified as Trichoderma viride(Fig.15).

Conclusions and Discussion

The test results by confronting culture and growth rate method showed that different strains had different inhibiting effects on Cytospora chrysosperma. The inhibiting boundaries between T-33 colonies and pathogen colonies were clear, and T-33 strain prevent the spread of Cytospora chrysosperma toward itself.On the plates with poor inhibiting effects, the boundaries between two kinds of colonies were unobvious, and two kinds of mycelia extended toward and cover each other. The n-butanol extract of T-33 strain broth had the highest inhibiting rate (94%) against mycelial growth of Cytospora chrysosperma. This might be because that the n-butanol extract of T-33 strain broth destroyed the mycelial cell wall and membrane of pathogenic fungi,resulting in the leakage of intracellular materials, and further resulting in mycelial disintegration and protoplasm dissolution of Cytospora chrysosperma. It indicated that certain substances in the extract had stronger damaging effects on Cytospora chrysosperma. These kinds of substances might be both hydrolases[13]produced by Trichoderma during the culture (i.e. cell wall hydrolases and proteases) and allelochemicals[14]produced during the culture (i.e. antibiotics, chelating siderophores,lysozyme, bactericidal volatiles and detoxification enzymes). For example,the β-1, 3-glucanase produced by Paenibacillus could also degrade cell walls of Fusarium,resulting in mycelial autolysis and facture[15]. This might be caused by a combination of several mechanisms, and the specific inhibition mechanism is still needed to be studied further.

From the 29 Trichoderma strains,the highly efficient antifungal strain T-33 against Cytospora chrysosperma was screened, and the physiological and biochemical characteristics and culture characteristics of the T-33 strain were studied. For the growth of T-33 strain, the optimum carbon source was glucose with content of 3%(g/L); the optimum nitrogen source was soy flour with content of 0.02%(g/L);the optimum initial pH of medium was 6; the optimum culture temperature was 25 ℃. The T-33 strain can better absorb and utilize organic nitrogen sources, instead of inorganic nitrogen sources.The molecular biological analysis and blast analysis in the GenBank showed that the T-33 strain showed the highest similarity rate(99.9%) with Trichoderma viride; the full length of ITS sequence was 583 bp with GenBank accession number JF823649.

This study also found that the culture broth of Trichoderma viride strain T-33 contained certain types of antibacterial ingredients, which was consistent with the study results of Sun et al[16]. The verification test also showed that these types of antibacterial ingredients have thermal stability.So the T-33 strain has good development and application value. In future studies,the antibacterial ingredients in T-33 culture broth will be isolated, purified and identified, and the antibacterial mechanism will also be studied,thereby laying theoretical basis for the future development of green microbial pesticides.

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