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ISSN : 1225-9306(Print)
ISSN : 2288-0186(Online)
Korean Journal of Medicinal Crop Science Vol.24 No.5 pp.360-369
DOI : https://doi.org/10.7783/KJMCS.2016.24.5.360

Chemotactic Response Study of Cylindrocarpon destructans towards Ginseng Root Exudates

Yonghua Xu*, Kun Chi*, Aihua Zhang*, Fengjie Lei*, He Yang*, Yan Zhao*, Kuo Li*, Erhuan Wang*, Qiong Li*, Jong Seog Kim**, Seung Ho Lee***, Young Chang Kim***
*College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun 130118, China.
**Department of Rehabilitation, Woosuk University, Wanju 55338, Korea.
***Department of Herbal Crop Research, NIHHS, RDA, Eumseong 27709, Korea.
Corresponding author: +82-43-871-5506 (ycpiano@korea.kr)
June 6, 2016 July 11, 2016 September 28, 2016

Abstract

Background:

Cylindrocarpon destructans (Zins) Scholten is an important pathogenic fungus that causes ginseng root rot in many ginseng growing areas in China. Although C. destructans have been studied worldwide, research on its chemotaxis towards ginseng (Panax ginseng C. A. Meyer) root exudates in the rhizosphere remains limited.

Methods and Results:

In this study, we collected ginseng root exudates with three different polarities from three-year-old ginseng roots, and performed chemotaxis and spore germination assays to investigate the ability of these exudates to induce the response in C. destructans. The results showed that, compared with other conditions, when C. destructans cultivated at 20°C and a pH of 6 exhibited a strong positive chemotactic response toward 2 ㎎/ℓ aqueous phase, 20 ㎎/ℓ butanol phase, and 0.2 ㎎/ℓ petroleum ether from ginseng root exudates, the chemotactic moving indexes were 0.1581, 0.1638 and 0.1441, respectively. In addition, the spore germination rate with optimal chemotactic parameters were 48%, 53%, and 41% in the aqueous phase, butanol phase and petroleum ether groups, respectiviely, which were significantly higher than that in the control group (23%) (p < 0.05). The mycelial growth rate with optimal chemotactic parameters increased with culture time, and the maximum growth rates in the aqueous phase, butanol phase and petroleum ether groups were 0.425, 0.406 and 0.364 respectively, on the 4th day. The optimal chemotactic parameters were 39.73 ㎎/50 ㎎/ℓ , 48.93 ㎎/50 ㎎/ℓ , and 31.43 ㎎/50 ㎎/ℓ , in aqueous phase, butanol phase and petroleum ether respectively, from ginseng root exudates, compared with 5.5 ㎎/50 ㎎/ℓ , in the control group.

Conclusions:

The present study revealed that certain ginseng root exudates containing chemical attractants act as nutritional sources or signals for C. destructans and support its colonization of ginseng roots.


인삼 추출물에 의한 Cylindrocarpon destructans의 주화성 반응 연구

허 영화*, 시 곤*, 장 애화*, 뢰 봉걸*, 양 학*, 조 암*, 이 활*, 왕 이환*, 이 충*, 김 종석**, 이 승호***, 김 영창***
*중국 길림농업대학교 중약재학원
**우석대학교 재활학과
***농촌진흥청 국립원예특작과학원 인삼특작부

초록


    National Natural Science Foundation
    31100239
    © The Korean Society of Medicinal Crop Science. All rights reserved.

    This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    INTRODUCTION

    Panax ginseng C. A. Meyer (Ginseng, Araliaceae) in China and Korea is a kind of special pharmaceutical crops with great advantages and features, because the ginseng roots containing useful medicinal ingredients are highly valued, which are used world-widely to treat various diseases by herbal medicine practitioners (Jiao et al., 2014). However, a lot of factors such as deteriorated soil conditions (Dou et al., 1996; Huang et al., 1996), auto-toxicity (He et al., 2009), and plant diseases (soil sickness) would cause the continuous cropping obstacle of ginseng, which restricted the healthy growth of ginseng for long periods, and this also becomes a serious problem that restricts the development of ginseng industry. This study primarily focuses on plant diseases (Zhao, 1995).

    Because of the long-period plantation in the same area, soil-borne pathogens including bacteria, fungi, and nematodes get the opportunity to infect the ginseng root, which makes the ginseng plant vulnerable and low quality (Kim et al., 2006; Ohh et al., 1986). Among them, fungi are the major pathogens to cause ginseng root diseases, for example, ginseng rust rot disease caused by the infection of Cylindrocarpon destructans which is one of most popular and well known fungi. C. destructans usually affects the surface of ginseng root (Chung, 1975; Lee et al., 2015; Yu, 1987). Ginseng rust rot disease is widespread in many regions and countries with an incidence of 25% - 44%, or in even serious area, 80% - 100% to badly influence the production and value of ginseng root.

    It has been reported that the indirect activities of ecological effects of plant root exudates and imbalanced proportion of soil microbial population are considered to be the main factors to cause plant diseases. Some populations of microbes could make use of specific components of root exudates to achieve rapid growth by chemotactic response, thus inhibiting the growth of other beneficial microbial to change the composition and quantity of root exudates, which further provides more carbon and energy for the chemotactic and pathogenic microorganisms to create a vicious cycle resulting in stunting plant growth (Dixon et al., 1993; Zhou et al., 2010). Some report also said that chemotaxis may present a competitive advantage for certain detrimental microorganisms in early establishment on the root of many crop plants leading to reduce root vigor (Ling et al., 2011).

    Chemotaxis of specific microorganisms toward root exudates in soil is related to attraction toward specific components detected in the exudate (Li et al., 2014). During the time of ginseng growth, the ginseng root usually secretes many kinds of secondary metabolites, which are regarded as the specific components in the root exudates, and as it happens, the most productive period of secretion of ginseng root exudates are coincidentally the sameas the period of ginseng diseases outbreak. There is no relevant report showing whether exudates affect the outbreak of main soil-borne diseases and chemotactic response of major pathogens of ginseng, or which kinds of pathogens would be induced by the response, or what the relevant factors are, or what the mechanism is, or how to control the happening of plant diseases by exudates.

    Therefore, in this paper, three polarities (the aqueous phase, butanol phase and petroleum ether) of ginseng root exudates are used as experimental materials to research the chemotaxis response of C. destructans towards ginseng root exudates. This study will help us to understand the real process of energy exchange and information transfer between ginseng root and its surrounding soil environment, and to explore the important theoretical and practical significance to solve the problem of continuous cropping obstacle in ginseng.

    MATERIALS AND METHODS

    1.Microorganisms and microconidia

    The fungal strain C. destructans used throughout this study was isolated from the ginseng root in Panax ginseng of Ginseng Engineering Research Centre of Jilin, Jilin Agriculture University, Changchun, China. This strain was routinely incubated on potato dextrose agar (PDA) medium (Sigma-Aldrich Co., St. Louis, MO, USA) in petri dishes in the dark at 20℃ for 7 days and maintained at −80℃ in 30% glycerol for long-term storage. The conidial suspension of C. destructans was prepared according to Zhang et al. (2013). The conidia concentration was 1.0 × 106 CFU/㎖, which was determined by direct observation on the hemocytometer (Ningbo Hinotek Technology Co., Ltd., Ningbo, China).

    2.Preparation of experimental samples

    The aqueous phase, butanol (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) phase and petroleum ether (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) phase of three-year-old ginseng root exudates with the purity of 95% were obtained from Ginseng Engineering Research Centre of Jilin, Jilin Agriculture University, Changchun, China. The exudates samples of each polarity were rehydrated with sterilized Milli-Q water to 0.2, 2, 20 and 200㎎/ℓ respectively, which were filtered by mixed cellulose ester membrane filters (0.22 and 13㎜) (Whatman Co., Maidstone, England) to remove bacteria. The rose bengal medium (containing peptone 5%, KH2PO4 1%, MgSO4 0.5%, glucose 10%, chloromycetin 0.1%, bengal 0.033% and agar 18.5%) (Qingdao Hope Bio-Technology Co., Ltd., Qingdao, China) was used to cultivate the fungi during the experiments.

    3.Experiments of chemotaxis

    As shown in Fig. 1, a modified plate assay was used for quantitative chemotactic measurements, based on the materials described above (Morris et al., 1992). Briefly, three holes were punched by a hole puncher (Φ= 6㎜) in the rose bengal medium from top to down, which was orderly marked as A, B and C (A represents the test group with samples of ginseng root exudates, B represents solution of C. destructans spores, C represents the control group with sterile water) containing 30㎕ of corresponding solution in each hole. Subsequently, the distance between the three holes was 20㎜; A, B and C were connected with filter strips with the size of 25㎜ × 2㎜. Then, the assay were cultured at 25℃ for 4 days under dark conditions, and five replications were prepared for each treatment.

    4.Measurements of indicators

    1)Chemotactic migration index (CMI) and mycelial growth rate (MGR)

    The calculation standard of moving distance (calipers is used to measure the moving distance) was defined by the longest distance that the most hyphae could move, with a representing the moving distance of hyphae from B to A, b representing that of B to C; the CMI was a/b -1, the MGR was a -b/d (d represents cultivating days). The measurements of indicators were designed as follows in Table 1.

    2)Spore germination rate (SGR)

    Spore germination rate (SGR) was determined by the spore germination within one hundred spores observed through the microscope (10x eyepiece × 40x objective lens), and the SGR was calculated by N/100 in percent (N represents the number of spore germination in one hundred spores) (Li et al., 2009).

    3)Dry weight of mycelial (DWM)

    The mycelial growth weight was calculated by the formula; W2=W0-W1. W0 represents the weight of mycelia and filter paper after cultivating, W1 represents the weight of dried filter paper and W2 represents the mycelial growth weight.

    5.Experiments of chemotaxis of C. destructans towards ginseng root exudates in different concentrations, temperature and pH

    After filtering, three polarity ginseng root exudates with different concentrations (0.2, 2, 20 and 200㎎/ℓ) prepared experimental samples were used to conduct the experiments of chemotaxis as described above. On the basis of optimal concentration (2㎎/ℓ aqueous phase, 20㎎/ℓ butanol phase and 0.2㎎/ℓ petroleum ether), different temperatures (10, 15, 20 and 25℃) were investigated; on the basis of concentration and temperature (20℃), the 2㎎/ℓ aqueous phase, 20㎎/ℓ butanol phase and 0.2㎎/ℓ petroleum ether ginseng root exudates were modulated into different pH (5, 6, 7 and 8) to research the chemotaxis response of C. destructans under the temperature of 20℃. Five replications were used for each of the above treatment.

    6.Response of optimal parameters on the chemotaxis of C. destructans towards ginseng root exudates

    10㎕ spores suspension (1.0 × 106 CFU/㎖) was added respectively to the aqueous phase (2㎎/ℓ), butanol phase (20㎎/ℓ) and petroleum ether (0.2㎎/ℓ) ginseng root exudates on the microscope slides. Then slides were inverted in a moisturizing petri dish and cultivated in the dark at 25℃ for 12 h in the incubator. After that, the spore germination of one hundred spores were calculated by the microscope (10x eyepiece × 40x objective lens), and five replications were used for each treatment.

    7.Determination of the spore germination, mycelial growth rate and mycelial growth weight of C. destructans towards ginseng root exudates under the optimal parameters

    1)Spore germination

    10㎕ spores suspension (1.0 × 106 CFU/㎖) was added respectively to the aqueous phase (2㎎/ℓ), butanol phase (20㎎/ℓ) and petroleum ether (0.2㎎/ℓ) ginseng root exudates on the microscope slides. Then slides were inverted in a moisturizing petri dish and cultivated in the dark at 25℃ for 12 h in the incubator. After that, the spore germination of one hundred spores were calculated by the microscope (10x eyepiece × 40x objective lens), and five replications were used for each treatment.

    2)Mycelial growth rate

    Under the condition of the chemotactic optimal parameters, the hyphae were cultivated for different periods (2, 3 and 4 day) at the end of which, the mycelial growth rate (MGR) was determined and five replications were used for each treatment.

    3)Mycelial growth weight

    After the cultivation of ginseng root exudates for 7 days under the condition of the chemotactic optimal parameters, the cultivated mycelia were filtered by the quantitative filter paper with known weight (W1). Then, after the mycelium and filter paper were put into the oven for 2 h (80℃± 2℃), they were weighed (W0) again.

    8.Determination of newborn mycelial growth of C. destructans towards ginseng root exudates under the optimal parameters

    Digital microscope (10x eyepiece × 10x objective lens) was used to observe the newborn mycelial growth of C. destructans towards three polarity ginseng root exudates in the optimal chemotactic parameters on the third day of cultivation.

    9.Statistical analysis

    The differences among the experiments were analyzed via One-way ANOVA, followed by a least significant difference (LSD) test for each assay. All statistical analyses were performed with SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). In all statistical tests, p-values < 0.05 were considered statistically significance. All data are presented as means ± SD of five sample replicates.

    RESULTS

    1.Effect of different concentrations of ginseng root exudates on the chemotaxis response of C. destructans

    In the test, the fungus C. destructans showed varying degrees of chemotactic responses toward three polarity ginseng root exudates in different concentrations, but with the increasing concentration, all of them showed an ascending trend at first and then a descending one (Table 2). Among them, C. destructans exhibited the strongest positive chemotaxis responses toward ginseng root exudates in 2㎎/ℓ aqueous phase, 20㎎/ℓ butanol phase and 0.2㎎/ℓ petroleum ether, and the CMI was 0.1948, 0.1742 and 0.2024 respectively, which were significantly higher than the other groups (p < 0.05). However, when the fungus was cultivated in the ginseng root exudates of 200㎎/ℓ petroleum ether, it showed negative chemotaxis response.

    2.Effect of temperature on the chemotaxis response of C. destructans

    Under the cultivation conditions of different temperatures, C. destructans showed different chemotactic responses towards ginseng root exudates, and with the increasing temperature, the responses enhanced firstly and then receded (Table 2). Among them, with the optimal concentration, namely, 2㎎/ℓ aqueous phase, 20㎎/ℓ butanol phase and 0.2㎎/ℓ petroleum ether, C. destructans displayed the strongest positive chemotactic responses toward ginseng root exudates at the temperature of 20℃, and the CMI was 0.1411, 0.1425 and 0.1334 respectively, which was significantly higher than the other groups (p < 0.05). In addition, we found that the chemotactic responses under the temperature of 20℃ and 25℃ tests were higher than that of 10℃ and 15℃ tests.

    3.Effect of on the chemotaxis response of C. destructans

    It was found that when C. destructans was cultivated with ginseng root exudates of different pH, all the fungus showed positive chemotactic responses, and with the increasing pH, the responses presented strong at first and later became weak (Table 1). Among them, C. destructans exhibited the strongest positive chemotactic responses towards weak acid (pH = 6) ginseng root exudates in all three polarity, namely, aqueous phase, butanol phase and petroleum ether, and the CMI was 0.1734, 0.1667 and 0.1517 respectively, which was significantly higher than in the other groups (p < 0.05). Furthermore, the chemotactic responses under the condition of acid (pH = 5) and weak acid (pH = 6) were stronger than that of neutral (pH = 7) and alkaline (pH = 8). And the responses from the strongest to the weakest at the same pH (pH = 6) were aqueous phase, butanol phase and petroleum ether.

    4.Response of optimal parameters on the chemotaxis of C. destructans

    As shown in Fig. 2, C. destructans showed chemotactic responses towards three polarity ginseng root exudates in the optimal parameters, and the maximum CMI was 0.1581, 0.1638 and 0.1441, which appeared in the weak acid (pH = 6) ginseng root exudates of 2㎎/ℓ aqueous phase, 20㎎/ℓ butanol phase and 0.2㎎/ℓ petroleum ether in 20℃. Meanwhile, it indicated that there might be a link between chemotaxis response of C. destructans and the biological characteristics of fungus itself (Fan et al., 1995; Wang, 2001).

    5.Effect of optimal parameters of ginseng root exudates on the pathogen spores germination of C. destructans

    In the experiments, the spores of C. destructans germinated normally in the ginseng root exudates with three polarities under the optimal chemotactic parameters shown in Fig. 3. Among them, the spore germination rate was 48, 53 and 41% respectively in aqueous phase, butanol phase and petroleum ether groups, which was significantly higher than that in the control group (23%) (p < 0.05). The results implied that different compositions of ginseng root exudates not only induced the germination of pathogen spores, but also promoted the growth.

    6.Effect of optimal parameters of ginseng root exudates on the mycelial growth rate of C. destructans

    The fungi C. destructans was cultivated in three polarity ginseng root exudates (aqueous phase, butanol phase and petroleum ether) under the optimal chemotactic parameters. As the culture time went by, the growth rate of the mycelia was increased, and the maximum growth rates appeared on the 4th day when they were 0.425, 0.406 and 0.364 (Fig. 4) respectively.

    Interestingly, on the 2nd and 3rd day of cultivation, the speed of mycelial growth rate was that butanol phase group was the fastest, and then aqueous phase group and petroleum ether group, but on the 4th day, aqueous phase group was the fastest. Finally, by conducting the experiments, we found that the chemotactic responses caused by C. destructans could offer help to the selfgrowth and multiplication.Fig. 5

    7.Effect of optimal parameters of ginseng root exudates on the mycelial growth weight of C. destructans

    In spite that the mycelial weight of C. destructans was different among the ginseng root exudates of three polarities, they were dramatically still higher than that in control group. Furthermore, the mycelial weight of C. destructans in the control group was 5.5㎎/50㎖, but in the aqueous phase, butanol phase and petroleum ether test groups were 39.73, 48.93 and 31.43㎎/50㎖ respectively. Meanwhile, after filtering the culture solution, the number of C. destructans spores were 2.0, 2.75 and 1.75 × 107 CFU/㎖, respectively, in the aqueous phase, butanol phase and petroleum ether groups, compared with 5.0 × 105CFU/㎖ in the control group. Another phenomenon was that the of these filtered solutions was increased, because some nitrogenous substances of ginseng root exudates may be utilized by C. destructans as energy sources.

    8.Effect of optimal parameters on the newborn mycelial growth of C. destructans towards ginseng root exudates

    As shown in Fig. 6, the three groups of micrograph (A1 and B1, A2 and B2, A3 and B3) represented the edge density of the colony of new born mycelial growth of C. destructans towards ginseng root exudates, respectively. From the micrograph, we could see that when the newborn mycelia grown from center to both ends (Fig. 1), the mycelia grown in the test group was more intensive and massive with more branches, and the density was also higher than that in the control group due to the ginseng root exudates attraction. The results suggested that some compositions of ginseng root exudates may modulate the activity of new born mycelia and strengthen their capacity to transmit nutrients or moisture, and thereby induced the mycelia to accumulate nutrients for self-growth or multiplication.

    DISCUSSION

    The previous study has reported that organics produced by photosynthesis could be released into the rhizosphere as root exudates, some of which were all oleochemical and semio chemicals to provide carbon, energy and nutrition for microbial growth and multiplication. Meanwhile, some soil microorganisms with the help of chemotactic responses to the rhizosphere and root surface for colonization and reproduction. Different root exudates have certain influence in microbe species, microbial flora and microbial physiological characters, and the quantity of microbe is positively correlated with the root exudates accumulation (Badri and Vivanco, 2009; Bais et al., 2006). In the soil, pathogenic fungi are frequently affected by the root exudates which may attract or repel the nomadic pathogens, and may stimulate or inhibit the germination of resting vegetative forms. For example, some essential amino acids (arginine, lysine and histidine) secreted by mulberry sapling roots and flavonoids released by macadamias roots could promote conidia germination, mycelia growth and zoospores accumulation of some funguses (Liu et al., 2005; Zhenli and Zhiyi, 2005). The root exudates of leguminous plants contain some materials as signals to induce rhizobium to produce nodulation factors, which could, thereby, cause identification, infection, colonization and nodulation of rhizobium towards leguminous plants (Zhang and Zhang, 2005). However, few studies have reported the effects of ginseng root exudates on the chemotaxis of C. destructans, and ginseng rust rot disease is an urgent problem needing to be solved by ginseng-planting regions or countries (Lee et al., 2016).

    In China, the growth and development stage of ginseng lies in the period from late May to late June when root exudates gradually increase, reaching the peak period of ginseng rust rot disease (Wang, 2001). Though there are lots of factors that affect the occurrence of C. destructans, the exudates of roots definitely play a significant role in it. Our study accurately sought to analyze that the fungus exhibited stronger positive chemotactic responses towards the low and middle concentration of ginseng root exudates in aqueous phase, butanol phase and petroleum ether, and under the optimal chemotactic parameters, the spores germination, mycelial growth rate and dried weights of C. destructans significantly increased, whereas all of them decreased as the increase of root exudates concentration. Li et al. (2009) reported some of the ginseng root exudates inhibited the colony growths of C. destructrans at a high concentration and accelerated the growths at a medium concentration.

    Allelopathic effect on stress response can be dynamic and transformation, such as certain concentration can enhance allelopathic effect, but high concentration decreased allelopathy. It is because that the microorganisms converse between growth and defense by adjusting the external allelopathic material adaptability. Due to the presence of specific chemical receptor on the surface of the microorganisms membranes, it allows the microorganisms to detect changes in the concentration of chemical substances in the extracellular environment, and by intracellular delivery system will feel the transformation of chemical signals into intracellular signals and then effects the microorganisms taxis movement behavior.

    Nicol et al. (2003) found that ginsenosides secreted by American ginseng root had positive influence in the species composition and growth of the soil fungal (Phytophthora cactorum and C. destructans) community in the effective concentrations, while it inhibited the growth of harzianum. When Phytophthora megasperma infects soybean, its zoospores would be strongly attracted by the daidzein and genistein which are secreted in the exudates, and the chemotactic effect happens even in the low concentration, but when Phytophthora megasperma infects alfalfa and douglas fir, it does not show this kind of chemotactic response (Nicol et al., 2003). Bagga and Straney (2000) reported that apigenin could effectively stimulate the germination of Nectria haematococca spores, and such stimulation could take place in lower concentrations, but for other flavonoids, such as luteolin and hesperetin, this impact could be triggered in higher concentrations, and the effect of flavonoids as stimulation factors of spores germination was related with inhibition activity of cAMP phosphatase, thus increasing the ability of cAMP.

    Above all, we suggested that ginseng root exudates could be perceived, metabolized and transformed by the rhizo-fungus in the rhizosphere, and in addition, there might be an association between root exudates concentration effect and chemotactic response, colonization, reproduction, community building of soil microbial.

    Previous research has shown that the morbidity of C. destructans isolated in P. ginseng is higher in the roots before and after growth stage than that during the growth stage, while during three periods, there is no obvious difference in the external factors, and thus the rhizosphere environment plays an important role in ginseng root exudates. Zhang et al. (2014) has reported that root exudates contain ginsenoside, organic acid, phenolic acid, and other secondary metabolites, some of which would cause the acid-base imbalance of soil and lead to the chemotaxis attraction of some soil microorganism swimming or colonizing on the surface of plant root. Larsen et al. (2004) has reported that chemotaxis of some soil microorganism are sensitive to temperature, increased by the rising of temperature, which is related to methylation and phosphorylation of receptor proteins on the intracellular membrane of soil microorganism because the kinetics of enzymes that take part in the process of methylation and phosphorylation is influenced by the temperature.

    So we investigated the effect of and temperature of ginseng root exudates, which seems to be the important factors for chemotaxis of C. destructans. In this study, our findings suggested that when C. destructans was cultured in different and temperatures, the chemotaxis towards ginseng root exudates was different from that in rose bengal medium, i.e. it was higher in weak acid environment than that in the neutral and basic environment, meanwhile, chemotactic response was the highest at 20℃ compared with that at 10, 15 and 25℃.

    The study was not only indirectly verified that root exudates in the rhizosphere environment acted as specific chemoattractant for pathogenic fungus (C. destructans) growth and multiplication, but it also indicated that the chemotaxis of C. destructans towards ginseng root exudates may supply a positive advantage indetermining the outcome of its colonization.

    In this study, although three kinds of inducers couldn’t make C. destructans grew faster, they could release some volatile substances to forma gradient in the soil which could be detected by C. destructans, which led to the regulation of the mycelia activities, and the results were as follows; 1) Activities of mycelia on the side that receives ginseng root exudates was enhanced to promote the transmission capability of nutrition or water, thus the nutrition accumulating faster to reach a degree of saturation condition where more branches of mycelia grow to make the whole fungal colony look fluffier and firmer due to the repulsive interaction and larger angles of mycelia, 2) Another phenomenon was that ginseng root exudates may stimulate and resolve some intracellular molecules of C. destructans cells, and objectively improve the concentration of intracellular substances, which made the osmotic pressure of mycelia increase to absorb more water to grow fluffier and firmer. If there were any supportive materials such as membrane or medium around the mycelia, the mycelia easily touched upon the materials to grow along then, and however, if there were no such supportive materials, the growth of branches would be restricted, and as a result, the margin of bacterial colony would be on the decline. Thereby, from the macro view, the results were shown in Fig. 6 that mycelia grew toward ginseng root exudates, which formed the chemotactic response of C. destructans that we discovered. As for the formation mechanism, further studies are needed.

    ACKNOWLEDGMENTS

    This work was funded by the National Natural Science Foundation of China (project No. 31100239, No. 31200224, No. 31470420), the funded projects for Science and Technology Development Plan of Jinlin (project No. 20110926, No. 2011-Z25, No. 20130206030YY, No. 2014 0520159JH, No. 201410297) and the project supported by the Ministry of Science and Technology of China (project No. 2011BAI03B01). And, this work was carried out with the support of cooperative research program for Agriculture Science and Technology Development (PJ01018701), Rural Development Administration, Republic of Korea.

    Figure

    KJMCS-24-360_F1.gif
    The test operation map of chemotaxis response of C. destructans.
    KJMCS-24-360_F2.gif
    Response of optimal parameters on the chemotaxis of C. destructans towards ginseng root exudates.

    2㎎/ ℓ aqueous phase, 20㎎/ℓ butanol phase and 0.2㎎/ ℓ petroleum ether; both of the was 6 and cultivation temperature was 20℃. Vertical bars represent the standard errors of means from three experiments (n = 15).

    KJMCS-24-360_F3.gif
    Effect of optimal parameters of ginseng root exudates on the pathogen spores germination of C. destructans.

    ck; control group, sterile water. Vertical bars represent the standard errors of means from three experiments (n = 15).

    KJMCS-24-360_F4.gif
    Effect of optimal parameters of ginseng root exudates on the mycelial growth rate of C. destructans.

    ck; control group, sterile water. Vertical bars represent the standard errors of means from three experiments (n = 15).

    KJMCS-24-360_F5.gif
    Effect of optimal parameters of ginseng root exudates on the mycelial growth weight of C. destructans.

    ck; control group, sterile water. Vertical bars represent the standard errors of means from three experiments (n = 15).

    KJMCS-24-360_F6.gif
    The microscopic density growth map of mycelium colony edge of C. destructans which was affected by ginseng root exudates.

    Test group; A1; aqueous phase, A2; butanol phase, A3; petroleum ether. Control group; B1, B2 and B3. Magnification; 10x eyepiece × 10x objective lens, photograph of symmetry points.

    Table

    The measurements of indicators.

    Chemotactic response of C. destructans on the parameters (concentration, temperature and pH) towards ginseng root exudates (aqueous phase, butanol phase and petroleum ether).

    Different letters indicate that the values are significantly different at the 0.05 level with small letters (a - d) and 0.01 level with capital letters (A - D) by Duncan’s Multiple Range Test.

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