YIN Yue-lan, NIU Qi-chen, GAN Lu

(1. College of Animal Science and Technology, Yangzhou University, Yangzhou,Jiangsu Province 225009, China; 2 College of Grassland Science, Beijing Forestry University, Beijing 100083, China)

Abstract:Trichoderma is a promising biological control agent which can be used to control a variety of plant diseases,and it can also be used as a heavy metal adsorbent. In the study,83 strains of Trichoderma spp. covering eight varieties were collected from the grasslands in Beijing. Of these,8 strains were further evaluated for their antagonistic capacity against Clarireedia homoeocarpa which causes dollar spots in various turfgrasses. T. harzianum 152-22,T. brevicompactum 192-49,and T. virens 192-45 strains showed better inhibition of the pathogen due to their stronger competition for space and nutrients in dual culture plate,and the inhibition of secretive non-volatile and volatile substrates. Additionally,the cadmium tolerance of these strains was also investigated by the sequential gradual technique (0～350 mg·L-1). An increasing cadmium gradients in the culture medium inhibited the growth of Trichoderma,but T. virens 192-45 isolate was found to be more tolerant to cadmium toxicity and grow the best. Cadmium stress increased the sensitivity of the Trichoderma isolates to soil pathogen. In summary,T. virens 192-45 exhibited good tolerance of pathogen under cadmium stress in the grassland.

Key words:Grassland;Trichoderma; Clarireedia homoeocarpa; Biocontrol;Cadmium stress

Trichodermaspecies exist as soil fungi and are widely used as biocontrol agents and plant growth regulators in agriculture[1]. They also play an important role in the biodegradation of chemicals and various metals from wastewater and soil[2-3].

Trichodermahas long been considered a controlling factor of parasitic organisms.Trichoderma-based biocontrol is a complex mechanism,which is mediated by direct parasitism,production of antigenic metabolites and enzymes,competition for nutrients and rhizosphere occupation,induction of plant defense,promotion of plant growth,and resistance to stress in the environment[4-9]. As reported,Trichodermacan control the important plant diseases caused by phytopathogen,and commercialTrichoderma-based products have been widely used in biocontrol agents for controlling vegetable and crop diseases. However,there are few reports aboutTrichodermaon the biocontrol of lawn and pasture diseases. Lo et al[10-11]reported that commercialTrichodermaharzianum1295-22 strain can prevent turfgrass brown spots caused byRhizoctoniasolaniand dollar spots caused bySclerotiniahomoeocarpa.Trichodermaghanensestrain GCPL175 has a higher bactericidal capacity toLepiotastrain 1 506 causing mushroom ring in golf courses[12].Trichodermaprevents and controls diseases in grass,and improves the growth and biomass of turfgrass and pasture. For example,T.harzianumhad an obvious enhancement on the vegetative growth ofFestucarubraandPoapratensis[13]. Also,Trichodermacan be used as a biofertilizer to improve soil properties and microbial community structure and increase grassland biomass[14].

As a component of soil fungistasis,metal ions may influence the growth and antagonistic activity ofTrichoderma. It is well known that glucan and chitin,the main components ofTrichodermacell wall,could undergo redox reactions and adsorb with heavy metal ions[15]. However,there is also an accumulation process of active storage of heavy metals in living cells ofTrichoderma. For example,arsenic was removed by the adsorption of the cell wall ofT.asperellumSM-12F-1 and intracellular accumulation. In addition,for the ryegrass planted in Cd and Pb contaminated soil,the inoculation ofTrichodermasignificantly increased the total adsorption concentration of Cd and Pb in plants and the yield by improving its tolerance[16]. Therefore,the removal of heavy metals in the environment was the result of a variety of mechanisms,whether it is biosorption and accumulation or interaction with plants.

The occurrence of dollar spot caused byClarireediahomoeocarpa(formerly namedSclerotiniahomoeocarpa) has a devastating impact on turfgrass because it can infect almost all cold-season and warm-season turfgrass,such as creeping bentgrass (Agrostisstolonifera),bermudagrass (Cynodondactylon) and seashore paspalum (Paspalumvaginatum)[17]. Excessive use of fungicides on turfgrass and metal ions from wastewater can cause changes in overall biocontrol activities against plant pathogenic fungi. Thus,Trichodermaspp. has acquired an exceptional role as part of a sustainable approach to bioremediation of herbicide-laden lawns and soils.

Therefore,the present study was conducted to evaluate the potential of differentTrichodermaspp. isolated from the lawn in inhibiting the growth ofClarireediahomoeocarpaand the effect of resistance to Cd stress. Besides,a range of volatile and non-volatile compounds are produced by some species ofTrichodermaspp. which are active against pathogensinvitro. To determine the efficiency ofTrichodermaspp. againstC.homoeocarpaand Cd stress,strains were conducted with antagonistic and tolerability experiments in mediums where both can grow well in the laboratory. The present study was undertaken to find out respectively the most effective strain ofTrichodermaagainstC.homoeocarpaand Cd stress in turfgrass.

1 Materials and Methods

1.1 Isolation of Trichoderma and Clarireedia homoeocarpa from soil and turfgrass

The rhizosphere soil samples were collected from different locations of the grassland in Beijing,China.Trichodermastrains were isolated from soil samples (at 5～6 cm depth) by serial dilution technique,and then followed by plating 200 μL of each soil dilution onTrichodermaspecific medium (46 g potato-dextrose-agar,0.1 g streptomycin,0.3 g Chloramphenicol,0.02 g Rose Bengal sodium salt,1 000 mL distilled water) in a 9-cm-diam Petri plate at 25℃ in darkness. The probable colonies were observed closely and picked up from Petri plates and transferred to common potato-dextrose-agar (PDA) medium and finally pure culture was obtained by repeated subculture. Based on their morphological and molecular characteristics,they were identified at the species level.

Clarireediahomoeocarpastrains were individually collected from the leaves of creeping bentgrass infected by dollar spot,located at the putting green of Beijing Qinghe Bay Golf Course. The infected leaves and roots were soaked in 0.3% sodium hypochlorite for 10 minutes,then disinfected with 70% ethanol,and then thoroughly washed with sterile distilled water. They were transferred to PDA medium in Petri plates and incubated at 25℃ in darkness for seven days. The possible mycelia were closely observed and taken out of Petri dishes and transferred to PDA medium. Based on their morphology and the blast result of ITS sequence,it was identified asClarireediahomoeocarpa(GenBank accession number:MW455464).

1.2 Identification and Phylogenetic analyses of Trichoderma

DNA was extracted from the mycelial mat harvested from the surface of the broth with the Fungal Genomic DNA Extraction Kit (Aidlab Biotechnologies Co.,Ltd,Beijing,China). DNA sequences of nuclear internal transcribed spacers (ITS) rDNA regions (ITS1-5.8S-ITS2) and nuclear translation elongation factor 1 (TEF1) gene were amplified by PCR using the specific primers. The primers used were:ITS1 and ITS4;ef-728M and tef1r. PCR reactions used Taq Plus MasterMix (Dye) (CoWin Biosciences Co.,Ltd,Beijing,China) and PCR products were cleaned and sequenced. The sequencing results of allTrichodermastrains were done blast analysis and all sequences were submitted to GenBank (Accession number:MW454967-MW455050). MEGAX was used to align the sequences for subsequent analysis. Two pair phylogenetic analysis of ITS andTEF1 sequence matrix was computed using PhyloSuite with MrBayes mode and Maximum likelihood (ML) method[18].

1.3 Antagonistic efficacy of Trichoderma against C. homoeocarpa

Based on clustering analysis,ITS sequence similarity,and colony morphology,32Trichodermastrains were selected for the pre-experiment. Then,Trichodermastrains with one isolate whose inhibition efficiency higher than 80% were selected for the formal dual-culture experiment,includingT.longibrachiatum73-36,T.harzianum152-22,T.asperellum152-42,T.virens192-45,T.brevicompactum192-49,andT.atroviride252-30. The antagonistic efficacy ofTrichodermaisolates. againstC.homoeocarpainvitrowas evaluated on PDA medium. These were cultured in (28±1)℃ darkness for 6 days. Confrontation cultures were performed on PDA plates of 9 cm diameter. An agar block cut by a cork borer of 5 mm in diameter from the edge of the colonies. One each agar block including hyphae ofTrichodermaandC.homoeocarpawas placed in the center of the plate,5 cm apart from each other. At the same time,a plate receiving only the pathogen block was set as a control. For each treatment,three replicates were maintained and incubated at (28±1)℃ and darkness. After 3 days,it is measured that the colony radius of the treated pathogen and the control for calculating the inhibition rate. The formula for calculating inhibition rate is:inhibition rate/%= [(control radius-treated pathogen radius)/control radius]*100%.

1.4 Evaluation of antagonistic activity through production of antifungal non-volatile and volatile metabolites

For non-volatile metabolites,theTrichodermawas inoculated in the center of the PDA medium with sterilized cellophane (0.2 μm),and then cultured at (28±1)℃. Two days later,both cellophane andTrichodermawere removed. The tested pathogen was respectively inoculated in the original position and cultured at (28±1)℃ for 5 d. Meanwhile,the pathogen was inoculated on the PDA plate with no cellophane as control. The colony radius of pathogenic fungi on the plate was determined and the inhibition rate was calculated.

For the volatile metabolites,Trichodermaisolates andC.homoeocarpawere respectively inoculated in the center of the PDA medium by placing 5 mm block cut (5-day-old culture) from the edge of the actively growing region and incubated at 28℃. Two days later,the top cover of each petri dish inoculated withTrichodermawas replaced with the bottom of the same size culture medium containing the tested pathogen. The pairs of each plate were sealed with parafilm and incubated at (28±1)℃. PDA culture medium withoutTrichodermain the bottom of the culture dish and respective test pathogen covered on the plate were used as control. Each treatment was maintained three times. This combination was opened after 3 days,and the results of the observations were recorded by measuring the colony diameter of the test pathogen in each plate and the control plates.

1.5 Influence of cadmium on Trichoderma growth pattern

To understand the effect of cadmium on the growth ofTrichodermastrains isolated from grassland,32Trichodermastrains were selected for the treatment of sequential gradual cadmium concentration (50,100,150,200,250 mg·L-1) on PDA medium in the pre-experiment. That is to say,Trichodermawhose growth inhibition rate is less than 30% in PDA medium containing 50 mg·L-1cadmium was selected,and the fungus agar discs with this concentration were directly put into the next medium with 100 mg·L-1concentration,and so on to 250 mg·L-1. Finally,T.virens192-45 andT.harzianum152-22 were selected for the formal research objects.

The effects of cadmium toxicity on the growth pattern ofT.virens192-45 andT.harizianum152-22 were studied by culturing the fungus in PDA medium at eight cadmium concentrations (0,50,100,150,200,250,300,350 mg·L-1). Fungal agar discs as large as 5 mm were taken out from the colony edge of 1-week-old cultures,and inoculated at the center of a 9-cm-diameter Petri plate with PDA medium supplemented with different concentrations of cadmium. Three replicate plates were used for each treatment. The inoculated plates were incubated at 28℃ for 6 days in the dark,and the radial colony growth (mm) was measured daily. The percentage of inhibition (PI) in the radial colony growth after 6 days was calculated by the formula:PI/%= [(control radius - treated radius)/control radius] *100%.

2 Statistical analysis

The experiment was conducted with three replicates for measuring the radial colony growth. These data were statistically analyzed via the SPSS21.0 software,and subjected to a one-way analysis of variance (ANOVA) to determine the least significant difference (LSD) among different treatments atP<0.05 marked by different letters. For weight evaluation analysis,the technique for order preference by similarity to ideal solution (TOPSIS) method was used in R software.

3 Results

3.1 Identification and Phylogeny of Trichoderma spp.

A total of 83Trichodermastrains were obtained from 28 sites of grassland covered with different turfgrass. By annotating against the published sequences ofTrichodermain the National Center Biotechnology Information (NCBI),total isolates were identified as eightTrichodermaspecies. To identify our isolates accurately,the total sequences of ITS andTEF1 sequences were aligned with 11 taxa ofTrichodermaspp. from Genbank. The partition homogeneity tests of the combinations of the two gene regions were carried out,and theP-value of 0.001. Based on the tree topologies and aP-value of 0.001,the gene regions were combined in PhyloSuite software. The phylogenetic tree showed that theseTrichodermamainly separated into four clades. As shown in Figure 1,the first group included 20T.harzianumstrains,2T.afroharzianumstrains,1T.guizhouensestrains,and 2Trichodermaputative strains. In the next branch,11 isolates were divided into five subclasses,which were close toT.viride/virens. Besides,12Trichodermaisolates formed a branch closed withT.polysporum,and 36 isolates were grouped as a large branch withT.brevicompactum.

Fig.1 Phylogenetic analysis of all Trichoderma isolates using MEGAX and PhyloSuit with MrBayes and Maximum likelihood method on the basis of a combined ITS and TEF1 sequence matrix

Antagonistic activity ofTrichodermaagainstC.homoeocarpa

To determine whichTrichodermastrains had significant antagonist effects against pathogens,sixTrichodermaspecies with one isolate whose inhibitory effect was higher than 80% againstC.homoeocarpawere screened out for study.

As shown in
Table 1,Trichodermaspp. showed a significant reduction in terms of the radial diameter ofC.homoeocarpain confrontation cultures,in comparison with the control. Of these,T.asperellum152-42 strain showed a maximum extent of inhibition of 78.29%,followed byT.harzianum152-22 strain at 75.97%,T.virens192-45 strain at 69.38%. In the case of non-volatile metabolites,it was found that theT.virens192-45 strain reduced the growth ofC.homoeocarpaby 89.47%,and exceeded the above-mentioned isolates which showed antagonistic activity in the dual culture plate. Then,in the case of volatile metabolites,theT.harzianum152-22 isolate inhibited the mycelial growth of the tested pathogen by 43.96%,followed byT.atroviride252-32 strain with an inhibition rate of 40.94%. However,the volatile substances of all selectedTrichodermawere less effective in inhibiting pathogen growth than antagonistic experiment and putative non-volatile substrates.

Table 1 The mean inhibition of the mycelial growth (%) of the C. homoecarpa strains on the PDA medium through confrontation cultures,non-volatile and volatile effects under in vitro conditions,and analysis of weight evaluation and rank for inhibition in different modes

Inhibition values are mean±standard deviation. Different letters are statistically significant according to the LSD test (P< 0.05) by one-way analysis of variance (ANOVA) determination

To explore the inhibition effect of antagonism,non-volatile and volatile substances onC.homoeocarpa,the inhibition rate of three modes was integrated for the weight evaluation of variation by the TOPSIS method. By calculating the distance ratio between the positive and negative ideal samples,theT.harzianum152-22 strain had the greatest biocontrol potential againstC.homoeocarpa,followed byT.brevicompactum192-49 isolate andT.virens192-45 isolate (Table 1). Moreover,in the confrontation cultures,the common inhibitory band and the winding action on the hyphae of overlapping area betweenT.virens192-45 strain andC.homoeocarpawere observed (Figure 2).

Fig.2 The effect diagram of T. virens 192-45 against C. homoecarpa in the confrontation culture after 3 days,and the microscopic observation of inhibitory band in the overlapping area

3.3 The tolerance of Trichoderma from grassland to cadmium stress

During pre-experimentation,the aboveTrichodermaisolates were subjected to progressive cadmium stress,revealing twoTrichodermastrains (T.harizianum152-22 andT.virens192-45) that will perform better under the cadmium stress. We studied the growth pattern ofTrichodermastrains at eight cadmium concentrations to determine their tolerance to cadmium stress. The increase in cadmium concentration reduced the growth of bothTrichodermaspecies,however,the time-dependent growth response is different. ForT.harizianum152-22,within 1～4 days of treatment,the diameter of colonies placed on the culture medium with different cadmium concentrations was lower than that of the control group (Figure 3a). But,there are no significant differences between the control and 50 mg·L-1groups ofT.virens192-45 after 1 day of treatment. Furthermore,the diameter ofT.virens192-45 with the treatment of a concentration lower than 250 mg·L-1was almost the same as that of the control (Figure 3b).T.harizianum152-22 colonies treated on the 5th day reached a saturation state at the same time as the control colonies,and the concentration of cadmium reached its peak on this day is 100 mg·L-1. In line with this,the maximum threshold ofT.virens192-45 on the fifth day is 200 mg·L-1.

Additionally,the growth inhibition percentage ofT.virens192-45 andT.harizianum152-22 after 6 days treatment were also calculated for understanding the inhibitory effect of cadmium stress. ForT.harizianum152-22,when the concentration of cadmium in the medium increased to 300 and 350 mg·L-1,the growth of radial colonies was inhibited by 42.94% and 45.98% (Figure 3c). The growth inhibition rates ofT.virens192-45 are much better thanT.harizianum152-22 with an inhibition rate of 35.54% at 300 mg·L-1cadmium (Figure 3 d).

Fig.3 Growth pattern of T. harzianum 152-22 (a) and T. virens 192-45 (b) in response to cadmium concentration gradients in the PDA medium for 1～6 days. Percentage growth inhibition (PI) of T. harzianum 152-22 (c) and T. virens 192-45 (d) in response to cadmium concentration gradients in the PDA medium in 6-days. Values are mean of three replicates and vertical bars indicate±standard error. Different letters indicate statistically significant different according to LSD test (P<0.05)

4 Discussion

Trichodermais the most important component of the soil microorganisms and is genetically very diverse with several capabilities with agricultural and environmental significance,including attacking fungal pathogens,promoting plant growth,and being resistant to heavy metals[19]. Although biocontrol and soil remediation exist independently and have different mechanisms in the application ofTrichodermaspp.,they cannot be separated from the whole life process ofTrichoderma[20-21]. For example,the sugars in the primary metabolites produced byTrichodermaare related to antibiosis and inducing plant defense response,while the hydrophobin is related to the degradation of soil pollutants[22-24]. Besides,Trichodermacan also promote plant growth and the effectiveness of bioremediation[25]. In this study,the diversity and separation of theTrichodermacommunities were evaluated across grassland. Phylogenetic analysis and classification of 83 collectedTrichodermaisolates reveal that the abundance and diversity ofTrichodermaspecies were related to environmental factors. Then,a group ofTrichodermaisolates from diverse sample sites were identified as potential biocontrol agent that warrants further investigation.

There are various antagonism mechanisms ofTrichodermaagainst pathogens[26]. It is generally believed thatTrichodermahas competitive effects,producing antibiotics (including volatile and non-volatile substrates) and extracellular hydrolases,etc[27-28]. In the current research,the growth inhibition of the pathogens by fast-growing antagonists is more likely due to the competition mechanism for space and nutrients. The common inhibitory band was observed betweenTrichodermaand pathogen locked in plates (Figure 2),which indicates that further growth ofC.homoeocarpawas restricted byTrichoderma. Out of the six fungal antagonists studied for their efficacy,T.harzianum152-22,T.brevicompactum192-49, andT.virens192-45 had good antagonistic activity (Table 1).

Dollar spot (caused byClarireediaspp.) is one of the most important diseases of turfgrass which included creeping bentgrass,bermudagrass,etc.Clarireediahomoeocarpaprimarily infects leaves via mycelial growth and is distributed from a diseased leaf to a healthy leaf nearby[29,30]. In larger areas,the pathogen is distributed by physical means such as wind,rain,and human factors[30]. Although it does not infect roots directly,C.homoeocarpahas been associated with a root-damaging mycotoxin produced at a suitable temperature. AlthoughC.homoeocarpais a ‘nerve-wracking’ pathogen due to its fast growth speed and various modes of distribution,relevant studies[10]have shown that someTrichodermastrains significantly reduced dollar spot severity,such as commercialT.harzianumstrain 1295-22. The study also agreed with the point thatTrichodermaviresshad the strongest inhibition for the growth ofC.homoeocarpathrough three antagonistic approaches combined the competition for space and nutrients in plate culture with the inhibition of non-volatile and volatile substrates.

Trichodermawith combined resistance to heavy metals along with phytopathogens has the potential to promote plant growth and control pathogens in polluted soils[31],thus making it a preferred choice for both biocontrol and plant growth promotion[32-33]. The effects of cadmium concentration gradients on the growth ofTrichodermastrains were studied. To be efficient in this process,under a series of heavy metal stress conditions,the survivingTrichodermashowed tolerance to heavy metals through its growth pattern and speed. The results showed that the growth of mostTrichodermastrains was inhibited by the increase of cadmium concentration gradient in the culture medium suppressed,butT.virens192-45 andT.harzianum152-22 were tolerant to cadmium toxicity and might have cadmium bioaccumulation ability,especiallyT.virens192-45.

Conclusion

In general,thein-vitrostudy showed that the six strains ofTrichodermahave been antagonistic againstC.homoeocarpacausing turfgrass dollar spot. Among the sixTrichodermaspecies evaluated,T.harzianum152-22,T.brevicompactum192-49 andT.virens192-45 isolates showed significantly better performance. In addition,at most cadmium levels in the broth culture,it was calculated thatT.virens192-45 andT.harzianum152-22 had higher cadmium tolerance. Therefore,T.virens192-45 can be exploited as a potential biocontrol agent in cadmium-polluted lawns and agricultural fields. Although further field studies are necessary to test the efficacy of selected agents of biocontrol and bioremediation under field conditions and different management practices,this study establishes the potential of usingTrichodermastrains isolated from grassland for controlling turfgrass diseases and bioremediation in polluted soil.