Isolation, identification, and pathogenic effects of Trichoderma spp. from Auricularia auricula

Auricularia auricula, one of the most important edible mushrooms, is affected heavily by Trichoderma. We collected the diseased samples from the main A. auricula cultivation regions to characterize the pathogen and study the effect of Trichoderma spp. on A. auricula species. We identified one Trichoderma species, T. pleuroticola, based on the internal transcribed spacer and morphology characteristics, and two types of A. auricula strains, Heiwei 15 (HW 15) and Hei 29 (H 29), were tested in this work. The growth rate of T. pleuroticola was 3.26–3.52 times higher than that of A. auricula and advantageously competed for living space and nutrients. In confrontation culture, T. pleuroticola completely inhibited the mycelium growth of A. auricula and grew on it, resulting in a diverse impact on HW 15 and H 29. In addition, T. pleuroticola can produce metabolites with antibacterial activity. The inhibition rate of volatile metabolites to H-29 and HW 15 was 13.46% and 10.44%, and the inhibition rate of nonvolatile metabolites to H-29 and HW 15 was 36.04% and 31.49%, respectively. Further analysis showed that these antifungal activities inhibiting Auricularia auricula growth were mainly attributed to the organic compounds from T. pleuroticola, nonanal, tyrosine, beta-sitosterol, and wortmannin. In short, T. pleuroticola was a highly pathogenic fungi in the production of A. auricula. T. pleuroticola can affect the normal growth of A. auricula hypha, and its metabolites also have inhibitory effects on A. auricula hypha.

However, recent studies have shown that some Trichoderma strains can cause green mold disease of edible fungi, which is the most devastating disease. Massive attacks of the disease have been reported in South Korea, Sri Lanka, China, and Europe countries and pollute a variety of edible fungi, such as Agaricus bisporus, Lentinus edodes, Flammulina velutipes, and Auricularia auricula. Trichoderma spp. has caused serious losses in the production of edible fungi all over the world [5][6][7][8]. Studies have shown that Trichoderma can contaminate edible fungi strains, fruiting bodies, and compost. The contaminated edible fungus mycelium was brown, the fruiting body no longer grew, and there were green spores on the compost surface, which could not reproduce the fruiting body [9,10]. The antagonistic mechanism of Trichoderma includes indirect and direct mechanisms. The indirect mechanism competes for space and nutrients, while the direct mechanism is to parasitize fungi, produce active metabolites, and decompose enzymes [11]. The mycelium growth rate of Trichoderma is 1. 25-5.35 times that of L. edodes and P. ostreatus and can 1 3 96 Page 2 of 12 quickly occupy living space and compete for nutrients [12,13]. Trichoderma can parasitize in the mycelium of edible fungi. Studies have shown that T. harzianum and T. longibrachiatum cause the mycelium of edible fungi to swell, distort, dissolve, brown, wither, and die [14,15]. Trichoderma can secrete secondary toxic metabolites, extracellular enzymes, and volatile organic compounds to inhibit the edible fungi's growth, significantly reducing or even completely hindering commercial production [16]. Studies have shown that T. harzianum can produce extracellular chitinase to act on the cytoderm of P. ostreatus, leading to the disintegration of the cytoplasm [17]. There are many studies on the enzyme system of Trichoderma spp. and few on active metabolites. Only a few active substances have been identified, but the in-depth action mechanism is unclear.
Most of the studies on Trichoderma diseases of edible fungi are related to L. edodes, P. ostreatus, and A. bisporus, with less Auricularia auricula. This study collected a wealth of green mold disease logs from main A. auricula cultivation areas in Zhashui, Shaanxi, China. Based on the morphology and internal transcribed spacer (ITS) sequence, we analyzed the species of Trichoderma spp. To elaborate the mechanism of the Trichoderma spp., we undertook the Trichoderma spp. on the mycelial growth of A. auricula via a scanning electron microscope. In addition, we studied the volatile and nonvolatile metabolites of T. pleuroticola on the growth of A. auricula and speculated on the properties of antibacterial substances based on detecting the specific metabolites. This study provides an effective way to reduce or release the inhibition of T. pleuroticola metabolites on the growth of A. auricula and a reliable strategy for the safe production of A. auricula.

Sample collection and disease symptoms
Green mold disease occurred continuously in A. auricula production in Zhashui, Shaanxi, leading to a significant negative effect on the development of A. auricula. The diseased logs of A. auricula were collected from six main cultivation areas (see Table S1 in Supporting materials). The surface of the cultivation substrate was completely covered with Trichoderma spores, which became dark green, rotten, and soft, with the obvious musty smell. Hei 29 (H 29) and Heiwei 15 (HW 15) were sourced from the Institute of Microbiology (Heilongjiang, China).

Isolation and identification of Trichoderma strains
Trichoderma strains were isolated from green mold-affected logs of A. auricula. About 1 g of infected tissue of each sample was placed in a 250 mL flask containing 99 mL sterile distilled water and shaken for 20 min at 150 rpm. Samples were then serially diluted in sterile distilled water and transferred onto PDA. The plates were incubated at 28 °C. Pure subcultures were obtained from growing colonies on PDA.
The isolated species were incubated on PDA at 28 °C in darkness, during which colony shape and pigment were documented. Simultaneously, the cover glasses were inserted slantingly into the ACM medium to observe the conidia and conidiophores via the SEM (Quanta 200 Environmental Scanning Electron Microscope, FEI Company) when the mycelia spread on the cover glass. The strains were identified by Trichoderma Classification and Identification [18].
To identify Trichoderma spp., the growing mycelium on PDA plates was taken and DNA was extracted using the fungal genomic DNA kit. The amplification was made using the universal primer pairs ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′). The reaction mixture for PCR amplification was template DNA (1 μL), forward and reverse primers (1 μL), super mix (15 μL), and ddH 2 O (12 μL). The conditions of the thermocycler were as follows: 96 °C for 5 min; followed by 35 cycles of 96 °C for 20 s, 56 °C for 20 s and 72 °C for 30 s, and finally, 72 °C for 10 min. The amplicons obtained were confirmed in 1% agarose gel. PCR products were tested and sequenced by Beijing Genomics Institution. The obtained ITS sequences were submitted to the NCBI Gen-Bank (https:// www. ncbi. nlm. nih. gov/ genba nk/) and blasted in GenBank. Additionally, the phylogenetic trees were constructed with a neighbor-joining method by MEGA 7.0 [19].

Comparison of the mycelium growth rate of Trichoderma and A. auricula
T. pleuroticola was recovered from the Zhashui. T. pleuroticola and A. auricula (H 29 and HW 15) were selected as the test strains. The 5 mm diameter mycelial plugs were separately inoculated in the center of ACM and cultured at 28 °C until the mycelium had overgrown the Petri plate. Each treatment was replicated three times. The growth rate (cm/d) was calculated with formula (1).
where D is colony diameter (cm), 0.5 is plug diameter (cm), and T is cultivation days (d).

Effect of T. pleuroticola on A. auricula mycelium in Petri plates
To verify the interaction between T. pleuroticola and A. auricula (H29, HW15), a dual culture was used based on the Owaid method with slight modifications [20]. Mycelial agar plugs (5 mm in diameter) were cut from the growing front of 7-dayold colonies of A. auricula species. They were separately inoculated in ACM at 1 cm from the edge in Petri plates of 9 cm in diameter, and 5 mm mycelial plugs of T. pleuroticola were inoculated 6 cm apart on the same plates 7 d later. All combinations of T. pleuroticola × H 29 and T. pleuroticola × HW 15 were performed in triplicate. All plates were maintained at 28 °C in the dark, and the radial growth of A. auricula was measured every 12 h. The control group was plated with only the A. auricula. The confrontation inhibition of T. pleuroticola against A. auricula was calculated with formula (2). A cover glass was placed on the medium to promote mycelium climbing and observe mycelium interaction when the mycelium of T. pleuroticola and A. auricula were about to come into contact. After 24 h of incubation, the cover glass was removed, and then, the changes of A. auricula mycelium treated by T. pleuroticola were observed via SEM.
where R is the radial growth distance of the control plate (mm), R 1 is the radial growth distance of the test plate (mm), and P is the percent of inhibition (%).

Inhibition of T. pleuroticola volatile substances on A. auricula mycelium
The inhibition ability of T. pleuroticola volatile substances on A. auricula Mycelium was evaluated using two partition plate confrontation assays based on a modified Ebadzadsahrai et al. method [21]. Firstly, a 5 mm diameter plug of each A. auricula strain was inoculated on one side of the ACM plate and cultured for three days. Then, a 5 mm diameter plug of T. pleuroticola was inoculated on the opposite side. All plates were sealed with parafilm film and incubated at 28 °C. Radial growth of A. auricula was measured after three days. The control group was plated with only the A. auricula. Each treatment was replicated three times. The inhibition of T. pleuroticola volatile substances against A. auricula was calculated with formula (2).

Analysis of volatile organic compounds (VOCs) of T. pleuroticola
2 mL sterile ACM was placed in a 20 mL headspace bottle, and 5 mm diameter plug of T. pleuroticola after condensation was inoculated. All headspace bottles were incubated at 28 °C for 3 d, and the headspace bottle only contained 2 mL ACM as the blank control group (KB). VOCs were identified with gas chromatography-ion mobility spectroscopy (GC-IMS, FlavourSpec ® Gas Phase Ion Mobility Spectrometer, GAS Company, Germany). The test conditions were as follows: the headspace bath temperature was performed at 40 °C for 15 min; the temperature of headspace injection was performed at 85 °C, the injection volume was set at 500 μL, and the incubation speed was 50 rpm. The GC-IMS system was equipped with an MXT-5 (15 m × 0.53 mm and 1.0 µm) chromatographic column (RESTEK, USA). The chromatographic column was programmed to 40 °C and worked for 20 min. Nitrogen (99.999%) was used as the carrier gas and drift gas, and the flow rate of drift gas was 150 mL/min. The carrier gas was programmed to start at 2 mL/min (held for 2 min) and to linearly ramp up to 10 mL/min within 8 min, and then, it was linearly increased to 100 mL/min within 10 min. The temperature of IMS was 45 °C. All of the analyses were carried out in triplicate.  [22], T. pleuroticola filtrates in different culture periods were added to sterile ACM at 20% (v/v). A 5 mm diameter plug of each A. auricula was inoculated in the center of the plate. The A. auricula was placed on a fresh ACM plate as control. All dishes were incubated at 28 °C for 10 d, and then the radial growth of the A. auricula colonies was determined. The inhibitory activity of T. pleuroticola nonvolatile organic compounds against A. auricula was calculated with formula (2). Three replicates for each treatment were considered.

Analysis of nonvolatile organic compounds (nVOCs) of T. pleuroticola
To identify nVOCs, the T. pleuroticola filtrates were removed from the −80 °C refrigerator and thawed on an ice vortex for 10 s. Mix 50 μL of T. pleuroticola filtrates and 150 μL of 20% acetonitrile methanol internal standard extractant using the vortex of the mixture for 3 min and centrifuge (12,000 rpm, four °C) for 10 min. Then transfer 150 μL of the supernatant, and stand still at − 20 °C for 30 min. Finally, centrifugation (12,000 rpm, 4 °C) for 3 min followed by the analysis of the supernatant.
LIT and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (QTRAP), QTRAP ® LC-MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in positive and negative ion mode and controlled by Analyst 1.6.3 software (Sciex). ESI source conditions were set as follows: source temperature 500 °C; ion spray voltage (IS) 5500 V (positive), − 4500 V (negative); ion source gas I (GSI), gas II (GSII), curtain gas (CUR) set at 50, 50, and 25 psi, respectively; the collision gas (CAD) was high. The analysis was carried out six times.

Morphology identification of the isolates
One Trichoderma specie, KQQ-3, was isolated based on the colony shape, conidia, conidiophore, and pigment (Table S2 in Supporting materials). The color of the colony firstly is white round, with cotton-like aerial mycelium. The colony becomes dark green after 4 d because conidia clusters are generating. The mycelium grew all over the plate after 5 d, and the growth rate was 1.56 cm/d. The spores entirely covered the dish and arranged 2 ~ 4 concentric rings after 7

Molecular identification of the isolates
For further information, we applied the ITS sequences to identify the KQQ-3 accurately. The results suggested that the ITS sequence sizes of KQQ-3 were 606 bp (Fig. 2). The phylogenetic trees were constructed by the neighbor-joining method (Fig. 3) and demonstrated that the ITS sequence of KQQ-3 showed 99% identity with T. pleuroticola (JQ040377.1). Combined with microscopic characteristics, the agents causing green mold in the cultivation of A. auricula were identified as T. pleuroticola.

Comparison of Mycelium Growth Rate of T. pleuroticola and A. auricula
As shown in Tables S3, S4, and S5 in Supporting materials, the colony of H 29 and HW 15 were white rounds with short aerial mycelium, and the growth rate was slow, respectively, 0.56 cm/d and 0.52 cm/d, with no significant difference (p < 0.05). However, the growth rate of T. pleuroticola on ACM was 1.83 cm/d, which was 2.05 ~ 3.85 times that of A. auricula. It could achieve superiority in the living space occupation and its competition for nutrients.

Effect of T. pleuroticola on A. auricula Mycelium in Petri Plates
In dual culture, we observed the confrontation colony of two A. auricula species with T. pleuroticola and measured the inhibition rate for A. auricula mycelium (see Table S6 in Supporting materials, Fig. 4). T. pleuroticola inhibited heavily A. auricula mycelium growth; the inhibition is observed only on the 12th hour. After the prolonged confrontation, the inhibition ratio gradually increased to 100% after 60 h. From the perspective of mycelium morphology, T. pleuroticola mycelium could overgrow and spread on A. auricula mycelium, forming conidial clusters and resulting in the gradual withering of A. auricula mycelium. Besides, brown pigment appeared on the back of A. auricula. The phenomenon of the test was consistent with what Wang's group reported [7]. By analyzing the dark brown on the back of the mushroom colony, Li believed that the pigment produced by

edodes infected by
Trichoderma could produce dark brown vesicle rupture later. The contents infiltrated the medium, resulting in the dark brownness of the medium. At the same time, the top of Trichoderma mycelium in contact with L. edodes was dark yellow and secreted dark antibacterial substances to act on L. edodes mycelium, resulting in roughness and depression [24]. Marik et al. showed that the dark substance peptaibols secreted by T. pleuroticola, which inhibited the mycelium growth of A. bisporus, may be related to the dark brown color of the edible fungus colony [25]. It is speculated that T. pleuroticola and A. auricula may cause the formation dark brown color of the edible fungus colony in this test. However, a few studies on dark brown edible fungi are present in the literature, and many more studies should be performed to understand the production of dark substances. From the perspective of the mycelium interaction between T. pleuroticola on A. auricula, we observed that the mycelium untreated by T. pleuroticola was smooth and straight (Fig. 5a,  e). In contrast, the mycelium morphology of A. auricula from the interaction zone was abnormal. After contacting T. pleuroticola mycelium, H 29 mycelium became shrunken, broken, and dissolved (Fig. 5b-d). While HW 15 mycelium became swelled, broken, and dissolved ( Fig. 5f-h), it may be attributed to Trichoderma β-1,3-glucanase, chitinase, protease and other cell wall degrading enzymes. Additionally, there was no significant difference in the morphology of the different A. auricula strains treated by T. pleuroticola.
The mycelium interaction results were consistent with those Wu and Yan reported. However, the winding effect of Trichoderma was not found in this test. Wu thought that the mycelium twine only occurred in the L. edodes, which is very rare and was not the main reason Trichoderma infected edible fungi [15]. However, not all Trichoderma strains can inhibit the edible fungi by mycelium interaction. Innocenti et al. reported that T. pleuroticola and P. ostreatus mycelium grew in parallel during the confrontation, and the former inhibited the latter only by competing for space and nutrition [26].

Inhibition of T. pleuroticola volatile substances on A. auricula mycelium
We evaluated the inhibition of T. pleuroticola volatile substances. The results showed that the inhibition rate of T. pleuroticola to H 29 and HW 15 was 13.46% and 10.44%, respectively, with a significant difference (p < 0.05), and HW 15 had relatively strong resistance (Table 1). Only weak inhibition was observed; for the three days, the test was conducted. This may suppose that the inhibition of T. pleuroticola is not mainly due to the secretion of volatile substances or that volatile antibiotic substances are not generally produced if it is not approaching the A. auricula.

Analysis of volatile organic compounds (VOCs) of T. pleuroticola
To study the volatile substances of T. pleuroticola, GC-IMS was performed to analyze the samples. The results presented the information of each sample in the form of The qualitative and quantitative results of the volatile compounds in T. pleuroticola are shown in Fig. 7 and Table S7. GC-IMS detected 52 signal peaks and 40 typical compounds were identified, but there were still 12 compounds with no qualitative results due to the limited data of the library database. Since monomer ions and neutral molecules might form adjunct substances in the drift region, several single compounds might produce multiple signals so that the same substance could detect monomers or dimers. Based on the identified compounds, the volatile compounds in T. pleuroticola were alcohols (9.05%), aldehydes (14.33%), ketones (15.25%), furans (1.26%), and esters (45.20%). Among the compounds most identified in T. pleuroticola culture filtrate were ethyl acetate (31.52%), isopentyl alcohol (9.05%), acetone (8.03%), and butanal (4.93%).
The bioactive compounds detected in the culture of T. pleuroticola are members of the following compound classes: ethyl acetate could attract nematodes and collembolans, which nibble off fungus mycelium [27]; 2-heptanone has a penetrating fruity odor and is proven as an attractant for the bacterium Bacillus nematocidal lures nematodes to their death by a Trojan horse mechanism [28]; nonanol has various biological activities such as antimicrobial and antifungal activities, inhibiting sclerotia and ascospore germination, and mycelial growth of Sclerotinia sclerotiorum [29]; isoamyl acetate and isoamyl alcohol, the main Ginjo-flavour components of sake, had broad antimicrobial activity against filamentous fungi, bacteria, yeast, and Escherichia coli and Acetobacter aceti were markedly sensitive to them. Isoamyl acetate was hydrolyzed to acetic acid and isoamyl alcohol. Acetic acid and isoamyl alcohol had a high affinity for the cell membrane and abolished respiration in E.coli by damaging the cell membrane [30,31]. These volatile compounds may play a role in inhibiting the growth of A. auricula. However, we still do not know the mechanism of volatile compound inhibition. Furthermore, as the VOCs are complex mixtures and the environmental conditions induce their production, it is difficult to attribute the effects to individual volatile compounds or their mechanisms [32].

Effect of T. pleuroticola culture filtrate on A.
auricula mycelium

Inhibition of T. pleuroticola culture filtrate on A. auricula mycelium
In Table 2 and Fig. 8

Analysis of Nonvolatile Organic Compounds (nVOCs) of T. pleuroticola
In order to predict the T. pleuroticola metabolites with inhibiting A. auricula, the nVOCs of T. pleuroticola were used for metabolic profiling based on the widely targeted metabolomics approach. One thousand nine hundred seventy-three metabolites were detected and grouped into 15  (Table 3). T. pleuroticola was rich in metabolites belonging to the classes of amino acids and metabolites, organic acids and derivatives, coenzyme and vitamins, and nucleotide and metabolomics.
The analysis of nVOCs of the culture filtrates of T. pleuroticola tested led to the identification of 10 molecules in the literature ( Table 4). The results showed that tyrosine inhibited the growth of A. auricula. At the same time, threonine, glutamine, glycine, glutamic acid, phenylalanine, arginine, leucine, lysine, and methionine promoted the growth of A. auricula at low concentrations and inhibited the growth of A. auricula at high concentration [33]. In addition, wortmannin is an antibiotic and has remarkably specific antifungal properties, inhibiting the spore germination of Botrytis allii, Cladosporium herbarum, and Rhizopus stolonifera at the concentrations of 0.4-3.2 μg/mL [34]. Beta-sitosterol isolated from T. asperellum and T. harzianum showed inhibitory activity against Rhizoctonia solani, Sclerotinia rolfsii, and Fusarium oxysporum [35]. The secondary metabolites of Trichoderma sp. YM 311,505 Daidzein exhibited antibacterial activity against E. coli with a MIC value of 64 g/ mL [36].
However, a few studies on identifying nVOCs produced by T. pleuroticola are present in the literature. The studies understand that the antibacterial of the secondary metabolites produced is highly desired.

Conclusions
T. pleuroticola is a serious pathogenic fungus in the production of A. auricula. It captures more nutrients and space through faster growth and inhibits the growth of A. auricula mycelium through various interactions with mycelium. At the same time, T. pleuroticola secretes a variety of volatile and nonvolatile metabolites, including a variety of compounds with antifungal activities, which inhibit the growth of A. auricula separately or synergistically. In the future, we can not only use T. pleuroticola for the fermentation production of these active antifungal compounds but also carry out their chemical synthesis production on the basis of further studying the biosynthetic pathway of these active antifungal compounds in T. pleuroticola.
Author contribution BZ and ZW have designed this project and contributed to the main manuscript text. HD and QK conducted experiments and wrote the main manuscript text. XW, YL, HZ, YZ, XL, WW, and BBX have contributed to conducting the experiments, preparing figures, and writing. All authors reviewed the manuscript. ZW acknowledges the support from Oakland University.

Data availability
The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format, they are available from the corresponding author upon reasonable request.