Abstract Trichoderma asperellum is one of the species which can be isolated from contaminated Pleurotus ostreatus cultivation substrate with green mold disease. This study focused on the relationship between high temperature and infectivity of T. asperellum to P. ostreatus. Antagonism experiments between T. asperellum and P. ostreatus mycelia revealed that high temperature-treated P. ostreatus mycelia were more easily infected by T. asperellum and covered by conidia. Microscopic observation also showed that P. ostreatus mycelia treated with high temperature could adsorb more T. asperellum conidia. Furthermore, conidia obtained from T. asperellum mycelia grown at 36°C featured higher germination rate compared with that incubated at 28°C. High temperature-treated T. asperellum mycelia can produce conidia in shorter periods, and T. asperellum mycelia were less sensitive to high temperature than P. ostreatus. Deactivated P. ostreatus mycelia can induce T. asperellum cell wall-degrading enzymes (CWDEs) and P. ostreatus mycelia subjected to high temperature showed induced CWDEs more effective than those incubated at 28°C. Moreover, T. asperellum showed higher CWDEs activity at high temperature. In dual cultures, hydrogen peroxide (H 2 O 2 ) increased after 36°C, and high concentration of H 2 O 2 could significantly inhibit the growth of P. ostreatus mycelia. In summary, our findings indicated for the first time that high temperature can induce a series of mechanisms to enhance infection abilities of T. asperellum to P. ostreatus mycelia and to cause Pleurotus green mold disease.

Citation: Qiu Z, Wu X, Zhang J, Huang C (2017) High temperature enhances the ability of Trichoderma asperellum to infect Pleurotus ostreatus mycelia. PLoS ONE 12(10): e0187055. https://doi.org/10.1371/journal.pone.0187055 Editor: Sabrina Sarrocco, Universita degli Studi di Pisa, ITALY Received: March 6, 2017; Accepted: October 12, 2017; Published: October 26, 2017 Copyright: © 2017 Qiu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This research was financially supported by the National Basic Research Program of China (2014CB138303) and China Agriculture Research System (CARS20). Competing interests: The authors have declared that no competing interests exist.

Introduction Pleurotus ostreatus is one of the most important commercial edible mushroom crop in China. Its production is the third largest among all edible mushrooms in China. Pleurotus ostreatus is mainly cultivated using traditional agricultural cultivation methods in which production may be affected due to limited temperature control system. Green mold diseases caused by Trichoderma often occur during P. ostreatus cultivation [1]. These Trichoderma spp. mainly include T. pleurotum, T. pleuroticola, and T. harzianum [2,3]. As an emerging problem in P. ostreatus farms, green mold diseases have resulted in severe crop losses and huge economic losses [4]. Interestingly, extensive green mold diseases frequently occur when P. ostreatus mycelia have suffered from high temperature in summer during spawn running period. However, little is known about the causes why high temperature-treated P. ostreatus mycelia are more vulnerable to green molds. Therefore, it is of positive significance to find out the causes of green mold disease. Green mold diseases caused by Trichoderma are characterized by thick green sporulation on substrate surface [3]. Trichoderma conidia form quickly and profusely, and can quickly spread in the air due to their low weight. These floating conidia can be adsorbed onto fungal cell wall and recognized by lectin [5]. It was demonstrated that lectin activity plays an important role in host-mycoparasite interaction [6]. Conidia then germinate into mycelia, which quickly occupy nutritional sites and reproduce in mushroom substrate. Temperature is an important abiotic factor that affects Trichoderma growth and reproduction. Trichoderma conidia produced at high temperature exhibit maximum germination and growth [7,8]. Trichoderma spp. have been widely used to control plant pathogenic fungi and improve soil environment [9]. Such biological control includes competition for nutrients and space, production of volatile and nonvolatile antibiotics, and production of hydrolytic enzymes [10]. However, Trichoderma spp. are common pathogenic fungi for edible mushrooms [11]. Trichoderma spp. can grow on the mushroom beds and finally cover the entire substrate. Trichoderma features a variety of mechanisms that inhibit or degrade other fungal mycelia [12]. To infect other fungal cells, Trichoderma species should first recognize the host cell wall, coil around its mycelia, develop appressoria, and then secrete cell wall degrading enzymes (CWDEs) [13]. Trichoderma species can produce a wide variety of CWDEs, such as chitinases, β-1,3-glucanases and proteases to hydrolyze the main components of host cell wall during antagonism [14], which is a main mechanism for controlling other fungi [15]. In the presence of host fungi, production and enzyme activity of these CWDEs secreted by Trichoderma could be further induced [16]. Previous studies have focused on isolation and identification of pathogenic fungi causing Pleurotus green mold diseases [17,18]. Trichoderma asperellum has been reported as one of the species in Pleurotus substrates with green mold disease [3]. Studies showed that P. ostreatus substrates subjected to high temperature were more easily infected by Trichoderma during P. ostreatus cultivation. The aim of this study is to investigate the effects of high temperature on T. asperellum infecting P. ostreatus mycelium and find out the relationship between high temperature and rampant breakout of T. asperellum in P. ostreatus spawn running stage.

Materials and methods Strains, media and growth conditions Pleurotus ostreatus P89 (CCMSSC 00389) was provided by the China Center for Mushroom Spawn Standards and Control. Trichoderma asperellum T11 (ACCC 32725) was deposited at the Agricultural Culture Collection of China. The internal transcribed spacer region (ITS) sequence was amplified by the primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’), and translation elongation factor 1α (tef1) sequence was amplified by primers EF1-728F (5’-CATCGAGAAGTTCGAGAAGG-3’) and TEF-LLErev (5’-AACTTGCAGGCAATGTGG-3’) [3]. ITS and tef1 sequences of T. asperellum T11 were deposited in NCBI GenBank (KY368169 and MF049065) In order to verify that T. asperellum can cause Pleurotus green mold disease, Koch's Postulates were applied (S1 Fig). Ten pieces of P89 mycelial discs (5 mm diameter) were inoculated into cotton seed hull substrate and incubated at 28°C for 10 days. Afterward, P. ostreatus bags were respectively placed in incubators with different constant temperature at 28°C and 36°C for 2 days. Finally, five pieces of T11 discs were inoculated into the bags and incubated at 28°C. Each trail consisted of 5 bags. For enzyme assay, conidial suspension collected from strain T11 was inoculated into 250 mL Erlenmeyer flasks containing 100 mL of liquid minimal synthetic medium (MSM). One liter of MSM contained 5 g glucose, 0.2 g MgSO 4 ·7H 2 O, 0.9 g K 2 HPO 4 , 0.2 g KCl, 1.0 g NH 4 NO 3 , 0.002 g FeSO 4 ·7H 2 O, 0.002 g MnSO 4 , 0.002 g ZnSO 4 and distilled water [19]. For other experiments, T11 and P89 mycelia were cultured on potato dextrose agar (PDA; Difco-Becton Dickinson, Sparks, MD) and potato dextrose broth (PDB; Difco-Becton Dickinson, Sparks, MD). To cover the whole plate, mycelia of T11 and P89 were grown on PDA at 28°C for 3 and 7 days, respectively. Collection of deactivated P. ostreatus mycelia Fifteen pieces (a diameter of 5 mm punch) of P89 mycelial discs from solid medium were inoculated into 300 mL of PDB and incubated in a shaking incubator at 150 rpm and 28°C for 5 days. Then, the Erlenmeyer flasks were divided equally into four parts and incubated at 28°C, 32°C, 36°C and 40°C respectively for 2 days. Finally, mycelia were collected with filter paper (Whatman, Ф11), washed thrice with distilled water, dried under vacuum and grounded into powder [20]. Antagonism experiments between T. asperellum and P. ostreatus mycelia P89 mycelial discs (5 mm diameter) were placed at one side of PDA plates and incubated at 28°C for 3 days. Then, the plates were respectively placed in incubators with different constant temperatures of 28°C, 32°C, 36°C and 40°C for 2 days. Then, T11 mycelial discs were inoculated at the other side of plates and incubated at 28°C for 7 days [21,22]. The distance between two mycelial discs measured 5 cm. All tests were performed in triplicate. Agglutination reaction between T. asperellum conidia and P. ostreatus mycelia To examine agglutination between T. asperellum conidia and P. ostreatus mycelia, P89 was grown on a thin layer of PDA on microscope slides at 28°C for 3 days. Then, the plates were transferred to incubators with different temperature at 28°C and 36°C for 2 days. Conidial suspension (1.86×107 CFU/mL) in phosphate buffered saline (PBS) was used to observe agglutination reaction. Attachment of T11 conidia to P89 mycelia was observed in dual culture. Conidial suspension was poured over microscope slides with P89 mycelia. Reaction was carried at 28°C for 30 min. Then, the suspension was gently washed thrice with distilled water. Finally, attachment of T11 conidia to P89 mycelia was observed under light microscopes. An optical microscope (OLYMPUS DP70, Tokyo, Japan) was used for observations. The magnification was 400 times. Determination of T. asperellum conidial germination rate after high temperature treatment T11 disc was inoculated on PDA and incubated at 28°C for 10 days. Then, T11 plates with numerous of conidia were transferred to incubator at 36°C for various periods of time (2 h, 6 h, 12 h, 24 h, and 48 h). Conidia were harvested separately with SDW. Conidial numbers in suspension were determined using a hemocytometer and adjusted to appropriate concentration. Then, 100 μL of diluted conidium suspension was spread onto 2% water agar plates [23]. All plates were incubated at 28°C for 2 days, then the number of germinated conidia was recorded. Each treatment group consisted of three replicates. Conidium formation time after high temperature treatments for different times T11 discs were inoculated on the center of plates and incubated at 28°C for 3 days. Plates were incubated at 36°C for different times (2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, and 72 h) and then transferred to 28°C to observe formation of conidia. Conidium formation time was denoted as the time when the whole plate was covered with green conidia. Mycelial growth at different temperatures Both P89 and T11mycelial discs were inoculated on the center of PDA plates and incubated at 28°C. When colony diameter reached 1 cm, the plates were transferred into different incubators at 28°C, 32°C, 36°C, and 40°C, respectively. Then, colony diameter in each plate was recorded daily [24]. Enzymatic assays For enzymatic assays, T11 conidia were inoculated into 100 mL MSM and MSMP. The formula of MSMP was exactly the same as MSM except that 5 g glucose was replaced by 3 g glucose and 2 g deactivated P. ostreatus mycelia. To detect the effect of temperature on enzyme activity, T11 conidia (1.80×107 CFU/mL) were inoculated in MSM and MSMP (using deactivated P. ostreatus mycelia cultivated at 28°C), and cultures were incubated for 3 days at 28°C with shaking at 150 rpm. Then, flasks were transferred to rotary shakers (ZQLY-180F, Shanghai Zhichu Instrument Company Limited, Shanghai, China) with different temperature (28°C, 32°C, 36°C, and 40°C) for 2 days. Liquid culture was collected by centrifuging (Sigma 3K30, Osterode, German) at 6,000×g for 10 min and supernatants were used as crude enzyme solutions or stored at -80°C until assayed. To detect the effect of different substrates on enzyme activity, T11 conidia (1.58×107 CFU/mL) were inoculated in MSMP, which used deactivated P. ostreatus mycelia collected from cultures growing at different temperatures. Cultures were incubated for 3 days at 28°C with shaking at 150 rpm. Then, crude enzyme solutions were collected as above. Chitinase activity was determined by the method of dinitrosalicylic acid (DNS) [25]. The reaction mixture contained 0.1 mL of 1% (w/v) colloidal chitin in 10 mM phosphate buffer (pH 7.5) and 0.1 mL of crude enzyme solution. Colloidal chitin (1%) was prepared according to the method of Lee et al. [26]. First, reaction solution was mixed and incubated at 40°C for 30 min, reaction was stopped by boiling for 3 min, cooled to 25°C for 2 min and centrifuged (Sigma 3K30, Osterode, German) at 10,000×g for 5 min. The supernatant with 100 μL volume was mixed with equal volume of DNS, and heated to 100°C for 10 min. Absorbance was measured at 540 nm in Thermo Scientific Microplate Reader (Scientific Fluoroskan Ascent FL, Waltham, MA, USA). One unit (U) of enzyme activity was defined as the amount of enzyme to release 1μmol of N-acetyl-glucosamine in reaction mixture per min, whereas GlcNAc (Sigma) was used as the standard. Laminarin (Sigma) (0.75% w/v) in 50 mM sodium acetate buffer (pH 5.0) was used to determine β-1,3-glucanase activity [27]. The reaction mixture contained 20 μL laminarin solution and 10 μL of crude enzyme solution in a microplate. The mixture was incubated at 50°C for 10 min. Afterward, 100 μL of DNS reagent was added to mixture, heated to 95°C for 5 min, and then cooled to 25°C for 2 min. The reaction mixture (100 μL) was transferred to an enzyme-linked immunosorbent assay (ELISA) microplate and absorbance was measured at 540 nm in Thermo Scientific Microplate Reader. One unit (U) of enzyme activity was defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute. Protease activities were determined using casein as substrate and assayed according to the method of Satake et al. [28] with slight modification. The assay mixture contained 100 μL of crude enzyme solution and 200 μL of casein solution. Assay mixture was incubated at 37°C for 1 h. Subsequently, 400 μL of 5% trichloroacetic acid (TCA) was added to stop the reaction. The solution was centrifuged at 12,000×g for 5 min at 4°C. Absorbance of supernatant was measured at 280 nm using a UV-spectrophotometer (TU-1810, PERSEE, Beijing, China). One unit (U) of protease activity was defined as a 0.001 absorbance increase per minute in supernatant per milliliter of reaction mixture [29,30]. Detection of hydrogen peroxide accumulation in dual cultures of T. asperellum and P. ostreatus under high temperature To detect H 2 O 2 accumulation in antagonism between T. asperellum and P. ostreatus, T11 and P89 were evaluated in dual culture tests. P89 mycelia were inoculated into 250 mL Erlenmeyer flasks containing 100 mL PDB and incubated for 3 days at 28°C with shaking at 150 rpm. Then, 1 mL of T11 conidial suspension (1.69×107 CFU/mL) was inoculated into flasks containing P89 mycelia and incubated at 28°C for 1 day. Then, flasks were transferred to rotary shakers (ZQLY-180F, Shanghai Zhichu Instrument Company Limited, Shanghai, China) at 28°C and 36°C, respectively for different time (1 h, 2 h, 6 h, 12 h, 24 h and 48 h). PDB flasks inoculated with either T11 only or P89 only were used as controls at 28°C for 3 days. Extracellular fluid was collected by centrifugation for 5 min at 8,000 rpm. The H 2 O 2 content was determined using a Hydrogen Peroxide Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), following the manufacturer’s instructions. Absorbance was measured at 405 nm. Sensitivity of mycelia to H 2 O 2 High concentration of H 2 O 2 can inhibit the growth of mycelium. In order to assess sensitivity of T. asperellum and P. ostreatus mycelia to H 2 O 2 , P89 mycelial discs and T11 mycelial discs were inoculated on the center of PDA plates with different concentrations of H 2 O 2 and incubated at 28°C. When the majority of plates were occupied by mycelia, diameter and morphology of colonies were observed. Statistical analysis Analysis of variance (ANOVA) and Duncan’s multiple range tests (P ≤ 0.05) were applied to calculate and analyze data. Data were analyzed using SPSS version 20.0 software (SPSS Inc., Chicago, IL, USA). Error bars represent the standard deviation of three replicates.

Discussion Different Trichoderma species live in different environments, and different climatic conditions determine their distributions. Many Trichoderma species have been isolated from infected oyster mushroom substrates [31,32]. In our study, T. asperellum was one of the most common strains in green molded substrate. Optimal growth temperature for Pleurotus spp. mycelia measures 20–30°C, and mycelial growth will decline when temperature increases up to 30°C [33,34]. For Trichoderma spp., optimal temperature spans 27–30°C [35]. The present study provides a new viewpoint that high temperature may be an important factor to cause the green mold diseases. Other studies have observed antagonism by Trichoderma against other fungi [21,36]. In our study, T. asperellum can outgrow P. ostreatus mycelia, produce more conidia and significantly inhibit growth of P. ostreatus mycelia after they suffered from high temperature (Fig 1). In addition, P. ostreatus mycelia subjected to high temperature were infected by T. asperellum (S1 Fig). These phenomena may demonstrate that the resistance of P. ostreatus mycelia to T. asperellum severely decreased after high temperature treatment (36°C, 40°C) or the ability of T. asperellum to infect P. ostreatus mycelium enhanced. Mycoparasitism has been considered the central mechanism for Trichoderma infection in other fungi. Recognition is a prerequisite in inhibiting and hydrolyzing other fungal cells. Trichoderma asperellum attaches to other fungal cell with cell wall carbohydrates that bind to fungal lectins [37]. In the present study, we observed that P. ostreatus mycelia treated at 36°C for 2 days can adsorb a large number of conidia (Fig 2), and this result had not been observed before. This phenomenon suggests that high temperature may increase the lectin content of P. ostreatus cell wall, causing P. ostreatus mycelia to adsorb more conidia and be infected by T. asperellum. This result also explains why hot summer weather possibly particularly causes outbreaks of green mold disease. Trichoderma asperellum conidia will germinate to form filamentous hyphae after attaching to P. ostreatus mycelia. In our study, germination rate of conidia showed an increasing trend after high temperature-treatment for different time periods. Conidia collected at 36°C gave the greater germination in comparison with that at 28°C. Previous publications confirmed that Trichoderma conidia can accumulate intracellular polyols such as trehalose and mannitol under high temperatures. These substances can increase germination rate, enhance conidial bioactivity and tolerance to adverse environmental conditions [38,39]. We discovered that T. asperellum mycelia treated with high temperature (36°C) can produce conidia in shorter periods compared with that incubated at 28°C (Fig 4). Possibly, high temperature may accelerate the transition from T. asperellum mycelial vegetative growth to reproductive growth. Thus, high temperature can cause T. asperellum mycelia to produce a large number of conidia in a very short period of time and speed up the spread of conidia, resulting in a large area of affected by green mold disease. Temperature is an important environmental factor during mushroom mycelial growth. In the present study, the results showed that P. ostreatus mycelia were more sensitive than T. asperellum to high temperature. Growth of P. ostreatus mycelia was almost completely suppressed at 36°C (Fig 5). At the same time, mycelium growth of T. asperellum was significantly faster than that of P. ostreatus at 28°C, 32°C and 36°C. The rapid growth of T. asperellum is conductive to its occupation of nutritional sites and space [40]. During the mycoparasitism, extracellular CDWEs secreted by Trichoderma can destroy other fungal cell walls [41]. In the present study, CDWE activities produced in MSMP were higher than those produced in MSM (Fig 6), demonstrating that deactivated P. ostreatus cell can promote extracellular enzyme activity. Zhang et al. [16] also reported that deactivated Fusarium oxysporum mycelia were more effective in inducing production and activities of T. harzianum extracellular enzymes. We also noted that high temperature can enhance extracellular enzyme activities. The highest activities for β-1,3-glucanases were detected at 32°C, whereas those for chitinase and protease were observed at 40°C. Previous studies have also found that found that the optimum temperature for chitinase was 40°C [42,43]. These findings can explain that high temperature can enhance the ability of T. asperellum to hydrolyze P. ostreatus cell wall. At the same time, Trichoderma CWDEs are inducible by substrate [44]. In this study, we investigated the effects of different substrates on enzyme activity (Fig 7). The results showed that P. ostreatus mycelia harvested at high temperature can significantly induce enzyme activity. The findings are similar to those in a previous study, which showed that CWDEs are influenced by substrates [45,46]. According to the results, we infer that high temperature may change intracellular metabolites of P. ostreatus mycelia or cell wall components and these changes can induce T. asperellum extracellular enzyme activity. During antagonism, the fungal cell can induce synthesis of reactive oxygen species (ROS), such as hydrogen peroxide (H 2 O 2 ) and superoxide radicals (O 2 .-), during their direct confrontation by Trichoderma, and excessive ROS levels can cause cell death, damage cell membrane and increase membrane permeability, and cause electrolyte leakage [47–49]. In the present study, extremely low concentration of extracellular H 2 O 2 was detected in cultures with either T. asperellum only or P. ostreatus only; however, high concentration of H 2 O 2 accumulated in dual cultures, and high temperature can further promote the accumulation of H 2 O 2 which reached the highest at 36°C for 6 h. Furthermore, sensitivity of P. ostreatus mycelia to high concentration of H 2 O 2 was significantly higher than that of T. asperellum mycelia. Therefore, it is shown that T. asperellum can produce H 2 O 2 to inhibit and damage P. ostreatus mycelia in the process of mycoparasitism, and high temperature can further promote the accumulation of H 2 O 2 to enhance the ability of inhibition. To our knowledge, this study is the first to report the relationship between high temperature and rampant growth of Trichoderma. All present results show that high temperature is an important factor that enhances abilities of T. asperellum to infect P. ostreatus. This study provides theoretical basis for comprehensive understanding of the outbreak of green mold disease.

Supporting information S1 Fig. Infection of T. asperellum on P. ostreatus mycelia. a P. ostreatus bags were incubated at 28°C for 10 days. Then, T. asperellum discs were inoculated into bags and incubated at 28°C. P. ostreatus mycelia were not infected by T. asperellum. b P. ostreatus bags treated with 36°C for 2 days were infected by T. asperellum and covered with green conidia. https://doi.org/10.1371/journal.pone.0187055.s001 (TIF) S2 Fig. Mycelial growth rate on different concentrations of H 2 O 2 . Trichoderma asperellum and P. ostreatus mycelial discs were inoculated in the center of plates with different concentrations of H 2 O 2 . T: mycelial growth rate of T. asperellum. P: mycelial growth rate of P. ostreatus. Data were analyzed by Duncan’s ANOVA test. Error bars represent the standard deviation of three replicates. https://doi.org/10.1371/journal.pone.0187055.s002 (TIF)

Acknowledgments We would like to thank Dr. Mingwen Zhao (Nanjing Agricultural University, Nanjing, China) for critical comments on earlier drafts of this manuscript.