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2024 Volume 4
Article Contents
Abstract
Introduction
Materials and methods
Materials
Preparation of mixed surimi samples
Rheological characteristics of mixed surimi samples
Apparent viscosity
Dynamic rheological behavior
Strength of mixed surimi gels
Textural properties of mixed surimi gels
Cooking loss of mixed surimi gels
Low-field nuclear magnetic resonance (LF-NMR) of mixed surimi gels
Color difference of mixed surimi gels
Scanning electron microscopy (SEM) of mixed surimi gels
Thiobarbituric acid-reactive substances (TBARS) determination
Statistical analysis
Results and discussion
Rheological properties of surimi gels
Steady-state shear properties
Dynamic elastic properties during gelation
Strength of surimi gels
Textural properties of surimi gels
Cooking loss of mixed surimi gels
Color of surimi gels
Changes in moisture distribution of surimi gels
Scanning electron microscopy of surimi gels
Thiobarbituric acid-reactive substances (TBARS) determination
Conclusions
Author contributions
Data availability
Acknowledgments
Conflict of interest
Rights and permissions
References
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ARTICLE   Open Access    

Identification of two antagonistic fungi and antifungal activity analysis against anthracnose in tea plant (Camellia sinensis)

  • College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China

  • # Authors contributed equally: Lu Liu, Haonan Guan

More Information
  • Anthracnose, a prevalent disease affecting tea leaves, poses a significant threat to tea yield and quality. Current control measures predominantly rely on chemical pesticides, raising concerns over environmental contamination and pesticide residues. In exploring biological alternatives, 13 epiphytic fungi were isolated from healthy tea leaves, with nine demonstrating non-pathogenic characteristics. Through a plate confrontation test, strains exhibiting high antagonistic activity against anthracnose were identified, notably En10 and En12, which effectively suppressed the growth of tea plant anthracnose pathogens. Morphological and molecular analyses classified En10 as Aspergillus niger and En12 as Talaromyces. The application of spore suspensions of varying concentrations of En10 and En12 onto tea leaves revealed a significant reduction in anthracnose incidence. Notably, En12 exhibited the capacity to enhance the antioxidant potential of tea leaves. These findings underscore the potential of utilizing antagonistic fungi En10 and En12 for anthracnose control in tea plant, offering valuable insights and technical foundations for biological management strategies against this disease.

  • Introduction

    Surimi gel, known as 'concentrated myofibrillar protein'[1], is a kind of gel prepared by processing fish tissue according to fixed steps, such as rinsing, dehydration, and chopping, then adding a certain number of auxiliary materials for crushing, molding, heating and cooling. Salt-soluble myofibrillar protein (mainly myosin) of surimi denatures and unfolds after heating, and then re-crosslinks and polymerizes to form large protein aggregates[2], which is the internal mechanism of forming the gel structure. To enhance the texture and taste of surimi gel products, 2%−3% salt is added to promote the formation of the protein gel network structure and enhance the solubility and functional properties of the products[3,4]. Nonetheless, numerous studies have confirmed that excessive salt intake may result in risks of disease to human health, such as hypertension, and coronary heart disease[5]. Therefore, the development of low-salt surimi products will be widely focused on in future research.

    Recently, two strategies have been innovatively proposed to improve or maintain the gel properties of low-salt surimi products: one is to use exogenous additives or sodium salt substitutes[6−8], and the other is to exploit new processing technologies[9−11]. Glutamine transaminase (TGase) is the most effective surimi quality improver to enhance the functional properties of protein, which could catalyze acyl donors in proteins (γ-Hydroxylamine group) and acyl receptors (lysine residue, primary amine compound, etc.) undergo acyl transfer reaction to form cross-linked structures[12−14]. Previous studies by Jiang et al.[15] revealed that the cross-linking effect of TGase catalysis depends on the content and spatial distribution of available substrates. In addition, lysine, as an ideal exogenous additive, has been widely used in different meat protein gel systems, which can effectively improve the gel properties of low-salt surimi gel[16] and emulsified chicken sausage[17]. Many researchers have spent a lot of time to obtain higher-quality surimi gel products through the combination of two or more exogenous additives. Many scientists devoted themselves to obtaining higher quality surimi gel products through the combination of two or more exogenous additives. For example, Cao et al.[18] demonstrated that lysine (Lys) could bring about the dissociation of actin under the condition of the presence of TGase, promoting the cooking yield and gel properties of oxidative damaged MP. Similar findings reported by Cando et al.[19] indicated that Lys could induce the changes in protein structure, which were favorable terms to heighten the cross-linking effect of TG and improve the strength of surimi gel.

    Recently, the term ε-Poly-lysine (ε-PL) was followed with interest since it's a natural amino acid polymer produced by microbial fermentation, and was speculated that it has a similar molecular structure and potential effect to Lys[20]. ε-PL, which is generally composed of 25−30 lysine residues connected by amide bonds (by ε-Amino and α-Carboxyl group), was often used as an antibacterial agent for the preservation of meat products and aquatic products[21]. Li et al.[21] explored the effect of preservative coating with ε-PL on the quality of sea bass fillets during storage. It turned out that, ε-PL treatment evidently reduced the thiobarbituric acid and volatile base nitrogen values of bass slices during storage, inhibited the growth of microorganisms, and improved the water retention, texture, and flavor characteristics of the fish. A previous study by Cai et al.[20] proved the bacteriostatic and fresh-keeping effect of the composite coating of protein and sodium alginate on Japanese sea bass, and found that the composite coating had an effect on inhibiting the proliferation of sea bass microorganisms (Escherichia coli, lactic acid bacteria, yeast, etc.), fat oxidation, protein degradation, and nucleotide decomposition. As a natural cationic polypeptide, ε-PL has the advantages of no biological toxicity and low viscosity of aqueous solution. It is noteworthy that it can produce strong electrostatic adsorption with negatively charged amino acids[22]. Its effective modification of protein helps it fully interact with gel component molecules and groups[23], which may play a role in enhancing the texture and water retention of protein gel.

    Nevertheless, few reports are based on the addition of ε-PL to explore the influence mechanism on TGase-catalyzed surimi gel properties. The current study sought to shed light on the effects of different additions of ε-PL on TGase-induced cross-linking effect and the performance of composite surimi gel, to provide a theoretical basis and reference for further research and development of 'low salt', efficient and healthy surimi products.

    Materials and methods

    Materials

    Testing materials, including marine surimi, transglutaminase (TG, enzyme activity 100 IU/g), and ε-Polylysine (ε-PL, ≥ 99% purity) were supplied by Jiangsu Yiming Biological Technology Co., Ltd (Jiangsu, China). Egg white protein was purchased from Henan Wanbang Chemical Technology Co., Ltd (Henan, China). All other chemicals were from Shanghai Yuanye Biochemical Co., Ltd (Shanghai, China) and were at least analytical grade. The ingredients of the surimi gel are shown in Table 1.

    Table 1.  Surimi samples with different treatments.
    GroupsSurimi
    (g)    		TGase
    (w/w, %)    		Egg white
    Protein
    (w/w, %)    		ε-PL
    (w/w, %)    		NaCl
    (w/w, %)
    CK300−−−0.5
    TE3000.47.0−0.5
    TE + P13000.47.00.0050.5
    TE + P23000.47.00.010.5
    TE + P33000.47.00.020.5
    TE + P43000.47.00.040.5
    TE + P53000.47.00.060.5
     | Show Table
    DownLoad: CSV

    Preparation of mixed surimi samples

    The prepared block surimi was firstly put into the chopping machine (or tissue masher) and chopped for 2 min, then different mass fractions of ingredients were added, while the chopping time was extended to 5 min (the temperature should be controlled below 10 °C during this process). The exhausted surimi paste was poured into the special mold (50 mm × 20 mm), which was heated in two stages to form the heat-induced surimi gel (40 °C water bath for 40 min; 90 °C water bath for 20 min). Above prepared surimi gel underwent an ice water bath for 30 min and finally stored overnight at 4 °C for further use.

    Rheological characteristics of mixed surimi samples

    Mixed surimi samples with different treatments were determined under a Haake Mars 60 Rheometer (Thermo Fisher Scientific, Germany) with 35 mm stainless steel parallel plates referring to the method described by Cao et al.[18], and each determination was repeated three times. After centrifugation (1,000× g, 3 min) and 4 °C, the degassed surimi sol (~2 g) was equilibrated at 4 °C for 3 min before measurement.

    Apparent viscosity

    The shear stress of the mixed surimi sol was measured at shear rates between 0−100 s−1.

    Dynamic rheological behavior

    The rheological properties of the mixed surimi sols were measured in an oscillatory mode of CD-Auto Strain at 0.02% and 0.1 Hz frequency, respectively. The heating temperature range is set to 20~90 °C while the heating rate is 1 °C·min−1.

    Strength of mixed surimi gels

    The TA-XT Plus physical property analyzer (TA-XT Plus, Stable Micro Systems Ltd, Surrey, UK) was used to analyze the mixed surimi gel strength of each group of samples (n ≥ 3) at room temperature, and the cube-shaped surimi gel samples are measured through the P/0.5 probe. Referring to the method previously described by Fang et al.[24], the test parameters are as follows, pre-test and test rate (1 mm·s−1); rate after measurement (5 mm·s−1); depressing degree (30%); trigger force (5 g); data acquisition rate (400 p/s).

    Textural properties of mixed surimi gels

    Concerning the method of Jirawat et al.[25], texture profile analysis (TPA) was employed to record the hardness, elasticity, cohesion, chewiness, and resilience of mixed surimi gels. It is well known that TPA can explore the textural properties of food through the texture analyzer (TA-XT Plus, Stable Micro Systems Ltd, Surrey, UK) equipped with a P/75 probe to simulate human oral chewing action and obtain the texture characteristic values related to human sensory evaluation. The physical property parameter settings were as follows: downforce (5 g), compression degree (50%), pre-test speed, test speed, and post-test speed (1.0 mm·s−1).

    Cooking loss of mixed surimi gels

    The cooking loss of surimi gel under different treatments was determined referring to the protocol of Dong et al.[26]. The cooked surimi gel sample was instantly absorbed dry and weighed (W5). The cooking loss (CL) is calculated as follows:

    CL(g/100g)=(M1−M2)/M1×100 (1)

    Where M1, weight of the sample before cooking; M2, weight of the sample after cooking.

    Low-field nuclear magnetic resonance (LF-NMR) of mixed surimi gels

    The LF-NMR of the mixed surimi gel was detected by a PQ001-20-025V NMR analyzer (Niumag Analytical Instruments Co., Ltd, Suzhou, China) referring to the previous method conducted by Li et al.[27]. Relevant characteristic parameters were set as follows: sampling frequency (200 KHZ), echo time (0.3 ms), and cumulative number (8). Keeping the surimi gel sample (approximately 2 g) at room temperature for 30 min, they were carefully put into a cylindrical nuclear magnetic tube (15 mm in diameter), and finally, the relaxation time (T2) was recorded using the Carr Purcell Meiboom Gill (CPMG) pulse sequence.

    Color difference of mixed surimi gels

    CM-5 colorimeter (Konica Minolta Sensing, Inc., Tokyo, Japan) was used for surimi gel with different treatments following to the procedure of Wang et al.[28]. Several 1 cm thick slices were cut from surimi samples (n = 3) selected from different treatment groups, and the gel whiteness was calculated according to the following formula:

    Whiteness=100−√(100−L∗)2+a∗2+b∗2 (2)

    Scanning electron microscopy (SEM) of mixed surimi gels

    According to the previously described procedure by Gao et al.[29], the square-shaped surimi gels (4 mm × 4 mm × 4 mm) were immersed in 0.1 mol·L−1 phosphate buffer (pH 7.2) containing 2.5% (v/v) glutaraldehyde for 24 h. The above samples were washed using phosphate buffer (0.1 mol·L−1, pH 7.2) three times and then subsequently dehydrated in a series of alcohol solutions. The microstructures of the mixed surimi gels were imaged using an FEI Verios 460 SEM (FEI Inc., Hillsboro, OR, USA).

    Thiobarbituric acid-reactive substances (TBARS) determination

    The TBARS value was analyzed by the method described by Hu et al.[30] with slight modification. Thiobarbituric acid solution (1.5 mL) and 8.5 mL of trichloroacetic acid solution were added to the sample in turn, the mixture was bathed in water at 100 °C for 30 min. The supernatant was extracted and centrifuged twice (3,000× g, 5 min): (1) The original supernatant (5 mL) was taken and the same amount of chloroform for mixed centrifugation; (2) The second supernatant (3 mL) and petroleum ether (1.5 mL) were taken for mixed centrifugation. Finally, a small amount of lower liquid was taken to determine the absorbance (A532 nm). The final result was expressed in malondialdehyde equivalent (mg·kg−1).

    Statistical analysis

    All statistical analyses of data were investigated by statistical product and service solutions IBM SPSS Statistics version 23.0 (IBM SPSS Inc., Chicago, USA). The LSD all-pairwise multiple comparison method was used for the least significance analysis, and p < 0.05 was considered to indicate significance. The experimental data are expressed as mean ± standard deviation (SD) and plotted using Origin 2019 (Origin Lab, Northampton, MA, USA) software.

    Results and discussion

    Rheological properties of surimi gels

    Steady-state shear properties

    The steady-state shear flow curve can be used to characterize the interaction between proteins. The steady shear flow changes of apparent viscosity (Pa·s) of composite surimi with shear rate (s−1) under different treatments are shown in Fig. 1a. The apparent viscosity of all samples decreased significantly (p < 0.05) with increasing shear rate, exhibiting shear-thinning behavior[31]. In the range of shear rates from 0.1 to 100 s−1, the samples with TGase addition were always higher than the control, indicating that the induction of TGase enhanced the cross-linking of surimi proteins and formed a more stable structure. The surimi samples added with a lower proportion of ε-PL (0.005% and 0.01%) were regarded as lower viscosity fluid (Fig. 1a). The possible reason was that the low concentration of ε-PL provided a weak effect on the pH value of the surimi system. As a consequence, the content of the net charge provided was relatively small and the electrostatic interaction between the surimi proteins was weakened[20].

    Figure 1.  Effect of different treatments on rheological properties of surimi.
    DownLoad: Full-Size Img PowerPoint
    Dynamic elastic properties during gelation

    The elastic modulus (G′) mainly depends on the interaction between protein molecules, which can reflect the change of elasticity in the heat-induced surimi gel. As shown in Fig. 1b, the gel process of surimi in the control group was a thermodynamic process consisting of two typical stages. In the first stage, the value of G' showed a gradual downward trend in the range of 20~50 °C. This is mainly because, (1) the activity of endogenous protease in surimi is increased; (2) the myosin light chain subunits of surimi are dissociated under the action of protease, forming myosin and actin[24]; (3) heat-induced hydrogen bonds break between protein molecules, thus enhancing the fluidity of the gel system[31]. The second stage: From 50 to 73 °C, the G' value increases rapidly. As the temperature continues to rise to 90 °C, the G 'value keeps rising steadily. At this time, the gel network structure gradually became stable and irreversible.

    Compared with several curves in Fig. 1b, TGase treatment could significantly increase the G' value. The change trend of G' value of the samples added with TGase and ε-PL were similar to that of the control group, but the former increased faster and the maximum value of G' was also significantly higher. The temperature corresponding to the first peak of G' gradually decreased (PL1≈PL2 < PL4≈TE < PL3≈PL5 < CK), indicating that the process of protein denaturation and aggregation was advanced. This result showed that the proper amount of ε-PL (0.04%) in combination with TGase can reduce the thermal denaturation temperature of protein-forming gel and improve the forming ability of composite gel, which was unanimous with the changes in texture and properties of surimi gel (Table 2). Furthermore, the addition of a small amount (0.005%) or an excessive amount (0.06%) of ε-PL made the mixed surimi protein system more unstable, affecting the ability of TG-induced gel formation as the last resort[32].

    Table 2.  Effect of different treatments on the textural properties of surimi gels.
    GroupsHardness (g)SpringinessCohesivenessChewiness (g)Resilience
    CK811.40 ± 45.28de0.81 ± 0.01a0.53 ± 0.02b498.36 ± 21.58c0.21 ± 0.01e
    TE1105.80 ± 61.83c0.83 ± 0.01a0.56 ± 0.01b510.40 ± 26.04c0.24 ± 0.00bcd
    TE + P1736.01 ± 18.49e0.80 ± 0.03a0.61 ± 0.00a348.78 ± 18.36d0.23 ± 0.01cde
    TE + P2777.45 ± 39.61de0.80 ± 0.01a0.60 ± 0.02a307.03 ± 12.97e0.22 ± 0.00de
    TE + P3869.66 ± 45.82d0.83 ± 0.02a0.63 ± 0.01a485.06 ± 22.73c0.28 ± 0.00a
    TE + P41492.80 ± 77.13a0.83 ± 0.01a0.60 ± 0.01a721.74 ± 31.55a0.26 ± 0.01ab
    TE + P51314.60 ± 84.10b0.81 ± 0.00a0.60 ± 0.02a652.36 ± 30.24b0.25 ± 0.01bc
    Different lowercase letters in the same column indicated significant differences (p < 0.05).
     | Show Table
    DownLoad: CSV

    Strength of surimi gels

    Gel strength is one of the vital indicators to test the quality of surimi products, which directly affects the texture characteristics and sensory acceptance of the products. The strength of mixed surimi gel with TGase was significantly enhanced by 23.97% (p < 0.05) compared with the control group after heat-inducing. This enhancement is probably caused by the crosslinking promotion of TGase (Fig. 2a). A deeper explanation is that under the catalysis of TGase, the ε-amino group on lysine and the γ-amide group on glutamic acid residues undergo acylation reaction inside or between proteins, forming ε-(γ-Glutamyl)-lysine covalent cross-linking bond which promotes the production of the protein gel network[26].

    Figure 2.  (a) Catalytic reaction of glutamine transaminase and (b) effect of different treatments on gel strength and cooking loss of surimi gel. a−d/A−D: values with different lowercase letters indicate significant difference (p < 0.05).
    DownLoad: Full-Size Img PowerPoint

    On the basis of adding TGase, the strength of the mixed gel with ε-PL concentration ranging from 0.005% to 0.03% was lower than that of TE (Fig. 2b). It was speculated that the surimi sample with low concentration ε-PL was a fluid with lower viscosity, and the interaction between protein and water is strengthened, while the electrostatic interaction between proteins is further weakened[33]. This was also consistent with the change in rheological properties in Fig. 1a. Nevertheless, with the increase of ε-PL content to 0.04%, the gel strength of surimi gel significantly increased and reached the highest value (781.63 g·cm), which was about 21.03% higher than that of the TE group (p < 0.05) (Fig. 2b). The increase of gel strength in this process was obtained due to the following three possible reasons, (1) ε-PL is a cationic amino acid with positive charge, which can improve the pH value of protein or meat product system; (2) The interaction between the ε-amino group of ε-PL and the aromatic residue of protein, namely the cation-π interaction, can change the structure of meat protein[34]; (3) Protein (mixed surimi system) with high ε-amino group content, TGase has strong gel improving ability[35].

    Such synergy (ε-PL = 0.04%), as a comprehensive result of different impactors, is that after the surimi was mixed under the optimal ratio conditions, the synthesis of TGase enzyme catalysis, pH value shift, changes in protein and amino acid composition, etc., increased the strength of various forces, thus forming a denser stereoscopic network structure. Note that when the content of ε-PL reached 0.06%, the gel strength of the corresponding surimi gel decreased by 10% compared with 0.04%. Similarly, others reported the result that alkaline amino acids led to the reduction of the strength of the myosin gel of bighead carp[36].

    Textural properties of surimi gels

    TPA, also known as whole texture, is a comprehensive parameter that determines the sensory quality of surimi gel, including hardness, elasticity, cohesion, chewiness, and resilience[37]. As shown in Table 2, the addition of TGase significantly improved the texture properties of surimi gel (p < 0.05), which is related to TGase's ability to induce surimi protein to form more ε-(γ-Glu)-Lys covalent bond during heating[38] (Fig. 2a). Compared with the cross-linking induced by TGase alone, the gel hardness of the mixed surimi gel treated with ε-PL showed a similar change to the gel strength (Fig. 2b). As the concentration of ε-PL increased from 0.005% to 0.04%, the hardness of surimi gel gradually increased until it reached the maximum (1,492.80 g). At the same time, we also observed that the elasticity, cohesion, and chewiness of the composite gel reached the highest values with the 0.04% ε-PL, and these were higher than those of the TGase-only group. Similar to the findings of Ali et al.[39], the author found that the combination of ε-PL and beetroot extract can effectively replace nitrite, which produced a marked effect in maintaining the color of Frankfurt sausage and improving the texture and performance. However, the data we collated above (Table 2) also showed a clear phenomenon, that is, the addition of ε-PL with lower concentration (0.005%−0.02%) or highest concentration (0.06%) was not conducive to the combination with TGase to improve the texture characteristics of mixed surimi gel.

    Cooking loss of mixed surimi gels

    Cooking loss indicates the water holding capacity of heated surimi, which usually represents the stability of the three-dimensional network structure of surimi gel[40]. The 0.4% TGase significantly reduced the cooking loss of surimi samples (p < 0.05), which was 4.91% lower than that of the control group (Fig. 2b). When the addition of PL (combined with TGase) gradually increased from 0.005% to 0.04%, the cooking loss of surimi showed a decreasing trend and reached the minimum at 0.04%. The ε-PL-added surimi gel samples had higher cooking loss compared with that of only with TGase. Most notably, this may be due to ε-PL is able to further exert the cross-linking effect of TGase. A similar finding was reported in the study by Ma et al.[22], adding ε-PL is able to improve the solubility of myofibrillar proteins. Proteins with high solubility are more easily induced by TGase during heating, which aggravates the cross-linking between ε-PL-protein or protein-protein and forms a disordered gel network structure with relatively weaker water-holding capacity.

    Color of surimi gels

    As shown in Table 3, the high lightness (L*), low yellowness (b*), and high whiteness of surimi in the control group were relatively low, with values of 72.49, 10.42, and 70.56 respectively. After TGase was added to induce cross-linking, these values increased significantly (p < 0.05), which may be related to the photochromic effect of water molecules released from gel matrix under TGase-induced surimi protein cross-linking reported in the previous study[41]. Furthermore, on the basis of adding TGase, it was interesting to see that the L* and whiteness values of surimi gel first increased and then gradually decreased with the increase in ε-PL concentration. The influence of ε-PL combined with TGase on the whiteness of gel can be attributed to three reasons: (1) The addition of ε-PL can effectively increase the substrate content of TGase, and promote the cross-linking between proteins, so as to obtain a much more compact surimi gel structure, which affects the refractive index of light; (2) The water retention performance of the high surimi gel was significantly improved with the addition of ε-PL[42] (Fig. 2b), at this time, the L* value (positively related to the whiteness) value showed a decreasing trend with the decrease of the surface free water content of the gel sample; (3) With the increase of ε-PL concentration, some colored substances, formed due to the accelerated Maillard reaction rate during the preparation of gel, may have an adverse effect on the improvement of gel whiteness[43]. After adding TGase to induce cross-linking, although the whiteness value of composite surimi was significantly enhanced, ε-PL was not enough to further improve the whiteness value of final mixed surimi gel, and high concentration so far as to have a negative impact on the whiteness value.

    Table 3.  Effect of different treatments on the color of surimi gels.
    GroupsL*a*b*Whiteness
    CK72.49 ± 0.82e−0.92 ± 0.01e10.42 ± 0.33d70.56 ± 0.65d
    TE77.82 ± 0.34ab−0.23 ± 0.02c12.29 ± 0.29bc74.64 ± 0.26ab
    TE + P178.39 ± 0.40a−0.14 ± 0.01a12.46 ± 0.12ab75.05 ± 0.41a
    TE + P278.42 ± 0.43a−0.17 ± 0.0b12.46 ± 0.15ab75.07 ± 0.32a
    TE + P377.48 ± 0.16b−0.18 ± 0.02b12.37 ± 0.09abc74.31 ± 0.11b
    TE + P476.34 ± 0.05c−0.22 ± 0.01c12.73 ± 0.02a73.13 ± 0.04c
    TE + P575.48 ± 0.18d−0.26 ± 0.01d12.01 ± 0.14c72.70 ± 0.19c
    Different lowercase letters in the same column indicated significant differences (p < 0.05).
     | Show Table
    DownLoad: CSV

    Changes in moisture distribution of surimi gels

    The water distribution in surimi gel products was measured by LF-NMR[44], and the results were inverted to obtain the transverse relaxation time (T2), which reflects the strength of water fluidity[45]. There are four fitted wave peaks which are assigned to four different water distribution states and wave peak ranges (Fig. 3, Table 4): strong bound water T21 (0.1−1 ms), weak bound water T22 (1−10 ms), non-flowing water T23 (10−100 ms) and free water T24 (100−1,000 ms)[46]. At the same time, similar characteristics (the main peak is centered on T23) are possessed by the water distribution of all surimi gel samples. The addition of TGase increased the proportion of non-flowing water, accompanying the significant decrease in the content of free water (p < 0.05), compared with the control group. It proved that the addition of TGase brought this phenomenon, namely the bound water in surimi gel was partially transferred to the non-flowing, which also indicated the reduction of cooking loss of surimi samples induced by TGase (Fig. 2). On the basis of adding TGase, the low level of ε-PL (0.005%, 0.01% and 0.02%) inhibited the transition of free water in surimi gel to non-flowing water, in which the proportion of T23 decreased by 2.05%, 2.74%, and 1.42% respectively compared with the surimi gel only with TGase, while T24 increased by 15.76%, 14.17%, and 4.43% respectively. The proportion of P23 in the surimi gel sample (ε-PL = 0.04%) was equivalent to that of the TE group, implying that the addition of appropriate ε-PL was not directly connected with the changes in water distribution in surimi after TGase-induced cross-linking, and slight addition would have a negative impact, which was unanimous with the above research results of cooking loss (Fig. 2a).

    Figure 3.  Effect of different treatments on the transverse relaxation time T2 of surimi gel.
    DownLoad: Full-Size Img PowerPoint
    Table 4.  Effect of different treatments on the transverse relaxation time T2 of surimi gel.
    GroupsP21 (%)P22 (%)P23 (%)P24 (%)
    CK2.175 ± 0.186c1.198 ± 0.012e82.863 ± 0.691bc13.764 ± 0.091a
    TE1.938 ± 0.095d2.166 ± 0.043a85.144 ± 1.336a10.752 ± 0.033e
    TE + P12.705 ± 0.067b1.414 ± 0.009d83.435 ± 1.616b12.446 ± 0.105c
    TE + P23.005 ± 0.003a1.845 ± 0.056b82.874 ± 1.739bc12.276 ± 0.108c
    TE + P33.107 ± 0.063a1.715 ± 0.033c83.950 ± 1.587ab11.228 ± 0.086d
    TE + P41.770 ± 0.018d2.099 ± 0.059a85.021 ± 1.756a11.110 ± 0.144d
    TE + P53.121 ± 0.007a1.758 ± 0.107bc82.136 ± 1.091c12.985 ± 0.018b
    Different lowercase letters in the same column indicated significant differences (p < 0.05).
     | Show Table
    DownLoad: CSV

    Scanning electron microscopy of surimi gels

    The effect of different treatment methods on the microstructure of surimi gel was observed by SEM. As shown in Fig. 4, the microstructure of the control surimi gel (Fig. 4a) was observed to be enormously loose, uneven, and irregular, with large pores, which explained the reason why the control surimi gel had low strength and large cooking loss (Fig. 2b). In addition, the morphological properties of another group of surimi gel (TGase-induced) are appreciably different from the scanning electron micrograph mentioned above (Fig. 4b). Numerous compact and evenly distributed small pores were observed, instead of the loose structure in the control sample, which accounted for the fact that TGase could interact with proteins to form a more uniform and orderly three-dimensional network structure in the process of gel formation[47]. Moreover, similar findings were also reported in the previous studies of Dong et al.[26]. When treated with TGase and ε-PL (from 0.05% to 0.04%), the surface of the surimi gel sample gradually became flat through observation (Fig. 4f). We found that some smaller pores were formed and some larger pores were slightly supplemented visually. Remarkably, when the addition of ε-PL increased to 0.06% (Fig. 4g), the structure of surimi gel was not as uniform as before, and some large and irregular holes appeared obviously. This phenomenon might be related to moderate ε-PL, especially 0.02%, which can promote the cross-linking of TGase with protein and the aggregation/denaturation of different components, which agreed with the change of coagulation strength of surimi samples.

    Figure 4.  Effect of different treatments on the microstructure of surimi.
    DownLoad: Full-Size Img PowerPoint

    Thiobarbituric acid-reactive substances (TBARS) determination

    TBARS value, which can reflect the degree of lipid oxidation and rancidity of surimi, is an important indicator for the occurrence of oxidation[48]. Some substances in surimi, such as unsaturated fatty acids, will inevitably undergo oxidative decomposition to produce a large amount of malondialdehyde (MDA), the latter reacts easily with thiobarbituric acid (TBA) reagent to produce red compounds with maximum absorbance at 532 nm. As illustrated in Fig. 5, when TGase and a certain amount of PL (from 0.005% to 0.06%) were added, the TBARs value of surimi gels decreased to 2.03 and 1.67 mg/kg respectively, compared with the control group (2.26 mg/kg). This shows that the addition of both can significantly reduce the TBARS value of surimi gel (p < 0.05), considered to be a cue for the fat oxidation of surimi being inhibited. In addition, we also found that the antioxidant effect of PL was dose-dependent. The ε-PL can be chelated by ferrous ions along with the ability to scavenge free radicals like hydroxyl radicals, ensuring its antioxidant properties[33]. The ε-PL was prospectively considered as an antioxidant additive to prevent surimi protein from deteriorating induced by oxidation[49]. Similarly, making use of lysine or ε-PL as antioxidants in plant protein and meat protein has been widely reported[33, 50]. Fan et al.[42] confirmed that ε-PL addition has a significant effect on maintaining a high total phenolic content and vitamin C levels in fresh lettuce, which also shows that it has certain antioxidant activity.

    Figure 5.  Effect of different treatments on TBARS values of surimi gel. a−f: values with different lowercase letters indicate significant difference (p < 0.05).
    DownLoad: Full-Size Img PowerPoint

    Conclusions

    The addition of ε-PL at different concentrations had an apparent impact on the characteristics of TGase-induced mixed surimi gel, accompanied by the following situations: The 0.04% ε-PL provided a synergistic effect to promote the aggregation and crosslinking of surimi proteins induced by TGase; The rheology, LF-NMR and SEM results showed that the appropriate concentration (0.04%) of ε-PL apparently enhanced the initial apparent viscosity and elasticity of surimi samples, which was conducive to the formation of a more dense and uniform three-dimensional network structure, further limiting the flow of water in surimi and the exudation of hydrophilic substances; Simultaneously, the network strength was strengthened along with the texture properties of the mixed surimi gel. Furthermore, ε-PL had a strong ability to inhibit lipid oxidation in mixed surimi gel, showing a concentration dependence. When the content of ε-PL ran to lowest (0.005%−0.01%) or highest (0.6%), the improvement of the quality of surimi mixed gel was opposite to the above. The results show that ε-PL can be perceived as a prospective polyfunctional food additive to improve the texture properties, nutritional advantages, and product stability of surimi products.

  • The authors confirm contribution to the paper as follows: study conception and design: Cao Y, Xiong YL, Yuan F, Liang G; data collection: Liang G, Cao Y, Li Z, Liu M; analysis and interpretation of results: Li Z, Cao Y, Liang G, Liu Z, Liu M; draft manuscript preparation: Li Z, Cao Y, Liu ZL. All authors reviewed the results and approved the final version of the manuscript.

  • The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

    • Acknowledgments

    This work was financially supported by the Natural Science Basic Research Program of Shaanxi (No. 2023-JC-YB-146), the fund of Cultivation Project of Double First-Class Disciplines of Food Science and Engineering, Beijing Technology & Business University (No. BTBUKF202215), the Innovation Capability Support Plan of Shaanxi (No. 2023WGZJ-YB-27), the Agricultural Technology Research and Development Project of Xi'an Science and Technology Bureau (No. 22NYYF057), and Jiangsu Yiming Biological Technology Co., Ltd. in China.

  • The authors declare that they have no conflict of interest.

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  • Cite this article

    Liu L, Guan H, Jiao M, Ma Z, Bao Y, et al. 2024. Identification of two antagonistic fungi and antifungal activity analysis against anthracnose in tea plant (Camellia sinensis). Beverage Plant Research 4: e032 doi: 10.48130/bpr-0024-0020
    Liu L, Guan H, Jiao M, Ma Z, Bao Y, et al. 2024. Identification of two antagonistic fungi and antifungal activity analysis against anthracnose in tea plant (Camellia sinensis). Beverage Plant Research 4: e032 doi: 10.48130/bpr-0024-0020
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Identification of two antagonistic fungi and antifungal activity analysis against anthracnose in tea plant (Camellia sinensis)

  • College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China

  • Received: 22 March 2024
  • Revised: 28 April 2024
  • Accepted: 13 May 2024
  • Published online: 03 September 2024
Beverage Plant Research  4,  Article number: e032  (2024)  |  Cite this article

Abstract: Anthracnose, a prevalent disease affecting tea leaves, poses a significant threat to tea yield and quality. Current control measures predominantly rely on chemical pesticides, raising concerns over environmental contamination and pesticide residues. In exploring biological alternatives, 13 epiphytic fungi were isolated from healthy tea leaves, with nine demonstrating non-pathogenic characteristics. Through a plate confrontation test, strains exhibiting high antagonistic activity against anthracnose were identified, notably En10 and En12, which effectively suppressed the growth of tea plant anthracnose pathogens. Morphological and molecular analyses classified En10 as Aspergillus niger and En12 as Talaromyces. The application of spore suspensions of varying concentrations of En10 and En12 onto tea leaves revealed a significant reduction in anthracnose incidence. Notably, En12 exhibited the capacity to enhance the antioxidant potential of tea leaves. These findings underscore the potential of utilizing antagonistic fungi En10 and En12 for anthracnose control in tea plant, offering valuable insights and technical foundations for biological management strategies against this disease.

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    Introduction

    • Tea, a significant cash crop in China, faces a growing array of diseases due to the expanding cultivation areas. Being a monocultural perennial crop, tea plant thrive in warm, moist regions with consistent rainfall, creating an environment conducive to pathogen proliferation. Currently, over 400 types of pathogens have been identified in tea plant, predominantly fungi and bacteria, with viruses and algae being less prevalent[1]. Given that the leaf is the primary yield of the tea plant, the impact of leaf diseases holds greater importance. It has been observed that diseased leaves exhibit a significant reduction in polyphenols, catechins, and amino acids in comparison to healthy leaves[2]. Tea leaf diseases, including tea plant anthracnose, blister blight, grey blight, and red leaf spot, are prevalent in tea plantations[3]. Among these, anthracnose, the most widespread disease in tea gardens, is attributed to infection by the Deuteromycotina colletotrichum. After infection, leaves exhibit dark green water stains initially, followed by the gradual emergence of brown or reddish-brown spots. Subsequently, the affected areas transition to a grayish-white color. The diseased leaves become fragile and can easily break. Severe outbreaks can lead to extensive damage, directly impacting the quality and yield of spring tea the following year[4].

      Tea disease control methods encompass agricultural, chemical, and biological approaches. Agricultural control involves strategic fertilization, precise pruning, and systematic harvesting in tea gardens to manage diseases. Chemical agents remain the primary means of disease control due to their cost-effectiveness, broad applicability, and high efficacy. However, the excessive use of pesticides can lead to the development of resistance in diseases and pests, causing environmental pollution and leaving behind pesticide residues that pose a threat to human health. As living standards improve, there is a growing focus on the quality and safety of tea, the conservation of biodiversity in tea gardens, and the mitigation of environmental pollution. The use of biological control methods has become increasingly crucial for the sustainable growth of the tea industry, drawing attention due to its non-resistance and safety benefits. Notably, the investigation of pathogen antagonism has garnered escalating interest[5]. Lu et al.[6] identified 35 Trichoderma isolates from the rhizosphere soil of a healthy tea garden, with seven exhibiting antagonistic activity against the tea grey blight pathogen. Zhu et al.[7] isolated Bacillus amylolytica JT68 from the tea plant rhizosphere, which demonstrated a 50.73% inhibitory rate of volatile organic compounds against tea plant anthracnose. Moreover, JT68 exhibited inhibitory rates ranging from 70.0% to 93.2% against various plant pathogens including Magnaporthe oryzae, Colletotrichum higginsianum, and Verticillium dahliae. Currently, JT68 is utilized in the production of tea leaf fertilizer. Yang et al.[8] through the organization separation of tea grey blight disease in the separate identification of microorganisms, in addition to screening to pathogen Pseudopestalotiopsis theae, also to screen the inhibitory action to the strains of pathogenic bacteria of kc-6, identified as Bacillus amyloliquefaciens. In addition to inhibiting the growth of pathogenic bacteria, kc-6 also had a better inhibitory effect on Phomopsis vexans, Phytophthora melonis and Fusarium graminearum.

      In nature, plants are actually in a symbiotic state with microorganisms. Endophytes refer to the microorganisms residing in different plant organs and tissues, which do not induce disease in the plant temporarily[9]. Endophytes encompass epiphytic fungi, actinomycetes, and bacteria. They are widely distributed and have developed a mutually beneficial symbiotic relationship through long-term co-evolution with plants[10]. Endophytes play a crucial role in protecting plants against pathogenic fungi, bacteria, viruses, and pests, enhancing the host's stress resistance under adverse conditions. For instance, non-pathogenic foliar fungi in Populus trichocarpa have been shown to influence the severity of Melampsora rust disease[11]. Endophytes also can produce secondary metabolites that inhibit pathogen effects on plants, such as Trichoderma activating host defense pathways to mitigate disease impact[12]. Additionally, endophytes can also induce systemic acquired resistance (SAR) or systemic resistance (ISR) against pathogen invasion[13−15]. Some of them secrete low concentrations of metabolites to antagonize pathogens[16].They colonizing almost all plants and conferring higher resistance to colonized hosts compared to non-colonized plants[17].

      Current research on tea anthracnose primarily focuses on isolating and identifying pathogens, with limited studies on biological control methods and few antagonists utilized for managing this disease in tea plant. This study isolated and identified epiphytic fungi present in healthy tea leaves, screening for those with potent inhibitory effects. The findings offer a potential approach for biologically controlling tea plant anthracnose and serve as a valuable reference for developing biocontrol agents against this disease in tea plant.

    Materials and methods

      Plant material and reagents

    • One-year-old Camellia sinensis 'Longjing 43' cutting seedlings were acquired from the Tea Sci-tech Demonstration Base of Northwest A&F University in Xixiang, Shaanxi Province, China. The second and third leaves, which were healthy and mature on the new branches of the tea plant with the same leaf size, were selected for the experiment. The potato dextrose agar (PDA) medium was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

      Pathogenic fungi

    • In the field, tea leaves with brown dead, irregular water stains on the edges, and other typical symptoms of anthracnose were collected. Fresh diseased leaves of tea plant with typical diseased spots were selected, and four pieces of leaf tissue (4 mm2) at the junction were cut with sterilized scissors (at the junction of the healthy and diseased regions). The cut diseased leaf tissue was disinfected with 0.1% H2O2 surface for 2 min, then transferred to 75% ethanol for 20 s, and rinsed with sterilized water three consecutive times for 1 min each time. The leaves were placed on sterile paper and excess water was removed from the leaf tissue. The sterilized leaves were transferred to PDA medium in a super-clean workbench, with four pieces in each dish. After labeling, the leaves were placed in a 28 °C incubator for dark culture. After the fungal growth, the mycelium was selected for purification, and then the plant pathogenic fungal were obtained.

      DNA was extracted using the CTAB method (200 mM Tris-HCl, pH 8.0, 50 mM EDTA, 20 mM NaCl, and 1% SDS)[18,19] and internal transcribed spacer (ITS) region was amplified by ITS1/4 primers (F: 5'-TCCGTAGGTGAACCTGCGG-3', R: 5'-TCCTCCGCTTATTGATATGC-3'). The procedures used for the amplification of ITS sequences were as follows: predenaturation at 95 °C for 3 min, denaturation at 95 °C for 30 s, annealing at 52 °C for 45 s, extension at 72 °C for 90 s, extension at 72 °C for 10 min after 35 cycles. The size of the amplified fragment was detected by 1.5 μL PCR product in 1.5% agarose gel electrophoresis, which was purified and sequenced in a sangon (Shanghai, China). The sequences were blast in NCBI GenBank and were identified as Colletotrichum camelliae with 99% similarity.

      The fully unfolded 'Longjing 43' second and third leaves were selected with 75% ethanol for surface disinfection, scrubbed with sterile water and air dried naturally. The sterilized head was used to damage the leaves, avoiding the vein position. A hole punch with a diameter of 6 mm was used to punch holes at the edge of fresh mycelia. The mycelia pieces were inoculated on the damaged part of the leaves, and fresh PDA medium was used as the control. Each strain had at least six duplicates. The inoculated leaves were wrapped in plastic wrap and cultured in an artificial climate chamber (25 ± 2 °C; humidity above 70%). To verify whether the results satisfied Koch's rule, the infected leaves were isolated and re-identified to determine whether they were pre-inoculation strains[20].

      Purification and morphological observation of antagonistic fungus

    • Fungus with obvious antagonistic effects against pathogens on PDA medium were selected in the process of successive transfer culture. Single hyphal tips were transferred to a new PDA and cultured for 5 d at 25 °C in the dark. The size and morphology of fungal spores, hyphae, and other reproduction organs were observed with a microscope (Olympus BX51, Olympus Corporation, Monolith, Tokyo, Japan).

      Molecular identification of antagonistic fungi and phylogenetic construction

    • To identify the isolate potential antagonistic fungi, the DNA was extracted by CTAB method[18,19]. The specific primers of ITS1/4 was used to amplify the sequences. The ITS sequence was amplified according to the above PCR procedure, and the products were detected by electrophoresis and sent to the biological engineer for sequencing. All sequences from this study were submitted to GenBank. BLASTn algorithm was used to compare with the GenBank sequence database. According to the results of BLASTn analysis, the ITS sequences of the top ten species with the highest similarity were included in the phylogenetic analysis as references. Use MEGA5.0's default settings for its multiple sequences and manually optimize to achieve maximum sequence similarity. A phylogenetic tree was constructed based on the neighbor-joining method. Bootstrap analysis was carried out using the p-distance model. A Bootstrap value of 1,000 repetitions was used to assess clade stability and phylogenetic tree reliability.

      Antifungal activity analysis

      Dual culture tests

    • All fungal strains were transferred from stored cultures onto PDA plates and cultured at 25 °C in the dark. A mycelial plug (5 mm in diameter) of Colletotrichum camelliae cut from the margin of the medium area was placed on the center of a new PDA plate (9 cm in diameter). In the tested group, the potential antagonistic fungi were inoculated at a distance of 4 cm from the pathogenic fungi with two 5 mm mycelial plugs on its left and right side, while the corresponding site was replaced by two 5 mm PDA plugs in the control group. Each combination had at least three biological replicates. All plates were cultured at 25 °C in the dark and observed daily. When the pathogenic fungi stopped growing or the colony spread over the whole plant in the control group, the antagonistic type and the area of the pathogen colony were recorded and measured. If the tested fungi inhibited the growth of the pathogen and grew faster than the pathogen or the parasitized pathogen, the fungi was considered to have an antagonistic effect against the pathogen in vitro.

      Disease resistance experiments on tea leaves

    • The fungi with antagonistic effects in vitro were cultured in PDA medium in the dark at 25 °C. The mycelium disk with a diameter of 5 mm was penetrated from the edge of the actively growing colony. The disc was inoculated to potato glucose broth (PDB) and shake cultured (140 rpm) at 25 °C for 3 to 4 d, and spores were collected by centrifugation[21]. Then, the spore suspension was prepared to 104/ml, 105/ml, and 106/ml, respectively. Spore suspensions of different concentrations were sprayed on both sides of healthy tea leaves and incubated at 25 °C and 70% relative humidity for 48 h in the artificial climate chamber. The blank control (CK) was treated similarly, but sterile water was used instead of spore suspension. Each leaf was punctured with a sterile needle to create a symmetrical wound. Then, a 5 mm mycelial plug of pathogen was inoculated on the wound site at the back of the leaf. All leaves were cultured at 25 °C and 70% relative humidity for 48 h in the artificial climate chamber. The four time points of 1, 3, 5, and 7 d were observed, and the area of lesions was measured using ImageJ software.

      The determination of antioxidant enzymes' activity

    • The UV-1800 spectrophotometer (Shimadzu, Japan) was used to determine the activity of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) according to a previously described protocol[22]. In brief, the samples for the assessment of antioxidant enzymes, SOD, POD, and CAT were treated with nitrogen blue tetrazole, guaiacol, and hydrogen peroxide methods, respectively.

      Statistical analysis

    • Statistical analyses were performed using Excel and GraphPad Prism 9. The means and standard deviations of the data were calculated and statistically analyzed with analysis of t-test.

    Results

      Isolation of epiphytic fungi and their antagonistic activity against the anthracnose pathogen

    • To identify epiphytic fungi that exhibit antagonistic effects against tea plant anthracnose pathogens, epiphytic fungi from healthy tea plant leaves were initially isolated and purified. These fungi were then tested for pathogenicity and assessed for their antagonistic properties. The findings revealed that a total of 13 epiphytic fungi were purified, which belonged to various genera, including Cercospora, Cladosporium tenuissimum, Fusarium oxysporum, Nemania bipapillata, Phoma sp., Phytophthora, Trichoderma, Aspergillus niger, Macrocybe gigantea, and Talaromyces (Table 1). Among them, nine species were not pathogenic to tea leaves. We conducted plate antagonism experiments on these nine epiphytic fungi against the pathogen Colletotrichum camelliae, respectively. As a result, it was found that four of them exhibited antagonistic properties against pathogens (Fig. 1). Furthermore, En10 and En11 exhibit antagonistic behavior towards pathogens through types of substrate competition and mycoparasitism, whereas En12 and En13 exhibit antibiosis type (Table 1, Fig. 1). Therefore, the En10 and En12 strains were selected for further analysis due to their unique antagonistic characteristics. Through examination of colony and spore morphology (Fig. 2a & b) and phylogenetic identification (Fig. 2c), it was determined that En10 and En12 were affiliated with Aspergillus niger and Talaromyces, respectively. These results suggest that these two endophytic fungi may have potential as biocontrol agents against tea plant anthracnose pathogen.

      Table 1.  Information of endophytes isolated from tea plant.

      No. Cultivar Taxonomy Pathogenicity Antagonistic type
      En01 'Shaancha1' Cercospora No −
      En02 'Shaancha1' Cercospora Yes −
      En03 'Shaancha1' Cercospora Yes −
      En04 'Shaancha1' Cladosporium tenuissimum No −
      En05 'Shaancha1' Fusarium oxysporum Yes −
      En06 'Shaancha1' Nemania bipapillata Yes −
      En07 'Shaancha1' Phoma sp. No −
      En08 'Shaancha1' Phytophthora No −
      En09 'Shaancha1' Trichoderma No −
      En10 'Longjing 43' Aspergillus niger No AC
      En11 'Longjing 43' Macrocybe gigantea No AC
      En12 'Longjing 43' Talaromyces No B
      En13 'Longjing 43' Talaromyces No B
      In column of 'Antagonistic Type', A, B and C represent competition for substrate, antibiosis and mycoparasitism, respectively.

      Figure 1. 

      Plate antagonism experiments on endophytic fungi against the pathogen Colletotrichum camelliae. Scale bar = 1 cm. CK: Two 5 mm PDA plugs were used as controls.

      Figure 2. 

      Morphological and molecular identification of En10 and En12. (a) Front and reverse view of En10 colony on PDA after 9 d, displaying hyphae and conidia. (b) Front and reverse view of En12 colony on PDA after 9 d, showing hyphae and conidia. (c) Phylogenetic analysis based on sequences of the internal transcribed spacer between En10 and En12.

      Inhibition of colony growth of anthracnose pathogen by antagonistic fungi

    • To further verify the inhibition of antagonistic fungi against the anthracnose pathogen, a dual culture test was executed. The results demonstrated that the two antagonistic fungi had a significant inhibitory effect on the colony growth of the anthracnose pathogen. After a culture period of 9 d, En10 and En12 exhibited inhibitory rates of 59% and 44% on the growth of the anthracnose pathogen, respectively (Fig 3). These results further confirm the antagonistic effects of En10 and En12 against the tea plant anthracnose pathogen in vitro.

      Figure 3. 

      Inhibition of colony growth of anthracnose pathogen by antagonistic fungi En10 and En12. (a) Dual culture test on anta gonistic fungi against the anthracnose pathogen. CK: Two 5 mm PDA plugs were used as controls. Scale bar = 1 cm. (b), (c) Area change of pathogen under the condition of antagonistic culture. The data represent the average ± SD of biological repeats, ns represents no significant difference and * stands for significant difference analysis.

      Control effect of antagonistic fungi against anthracnose on tea leaf

    • To further elucidate the efficacy of antagonistic fungi in controlling anthracnose in tea leaves, we conducted resistance experiments on tea plant. Tea leaves were sprayed with spore suspensions of antagonistic fungi at three distinct concentrations, including 104/ml, 105/ml and 106/ml. However, it was observed that spraying high and medium concentrations of spore suspension followed by inoculation pathogen resulted in a significant number of tea leaf deaths. For instance, after spraying 106/ml concentration of En10 and En12, the mortality rate of tea leaves reached 14.29% and 66.70% on the third day, respectively, while the control group had a mortality rate of 0%. On the fifth day, the mortality rate of tea leaves reached 42.86% and 83.30%, respectively, while the control group had a mortality rate of 42.86% (data not shown). Therefore, the concentration of 104/ml spore suspension was chosen for treatment. The results of the study demonstrated a noteworthy reduction in the incidence rate of tea leaves that were treated with antagonistic fungi, as opposed to the control group. This suggests that the application of antagonistic fungi on tea leaf has the potential to inhibit tea plant anthracnose (Fig. 4a & b).

      Figure 4. 

      Determination of the control effect of antagonistic fungi against the anthracnose on tea leaves. (a) and (b) indicate the images and the diseased area of tea leaves treated by blank solvent, fungi spores of En10 and En12 after 1, 3, and 5 d, respectively. The data represent the average ± SD of biological repeats. * means significant difference (p ≤ 0.05); ** represents significant difference (p ≤ 0.01); ns indicates no significant difference.

      Activities of antioxidant enzymes after En10 and En12 treatment

    • The activities of antioxidant enzymes in tea leaves treated with En10 and En12 were measured. The results indicated a significant upregulation of SOD and POD enzyme activities following En12 treatment compared to the control (Fig. 5a & b). Particularly noteworthy was the 5.47-fold increase in SOD activity, while CAT enzyme activity remained unchanged (Fig. 5c). In contrast, although En10 exhibited notable resistance to the anthracnose pathogen, treatment of tea leaves with it did not result in significant alterations in the activity of associated antioxidant enzymes (Fig. 5). It is suggested that En12 may enhance the antioxidant response of tea leaves, increasing their resistance to anthracnose.

      Figure 5. 

      Detection of enzyme activity in tea leaves after spraying with antagonistic fungi. (a)−(c) indicates the SOD activity, POD activity and CAT activity, respectively. CK: sterile water treated. En10 (En12): antagonistic fungi spores treated. The data represent the average ± SD of three biological repeats. * stands for significant difference analysis and ns indicates no significant difference.

    Discussion

    • Anthracnose, a prevalent disease affecting tea plant worldwide, hinders plant growth and diminishes tea quality[2]. Current control methods rely heavily on chemical agents. However, increasing consumer demand for healthier products has highlighted concerns over pesticide residues in tea and environmental contamination in tea gardens. The emerging field of endophytes in plant defense against pathogens presents promising alternatives for combating fungal, bacterial, viral, and pest-related threats in tea cultivation[17]. The potential application of endophytic antagonism in managing tea plant diseases is promising due to the abundance of epiphytic fungi in tea plant. Therefore, researchers have isolated and identified endophytes from the tea plant. So far, endophytes known to exhibit antagonistic activity against anthracnose include Bacillus amylolyticus[7], Bacillus subtilis[23], Bacillus atrophaeus[24], Bacillus velezensis[25] and Streptomyces luteosporeus[26]. In this study, epiphytic fungi were isolated from the leaves of healthy tea leaves, resulting in the purification of 13 epiphytic fungi representing Cercospora, Cladosporium tenuissimum, Fusarium oxysporum, Nemania bipapillata, Phoma sp., Phytophthora, Trichoderma, Aspergillus niger, Macrocybe gigantea, and Talaromyces (Table 1). Notably, nine of these species were found to be non-pathogenic to tea leaves. Experimental findings demonstrated significant antagonistic effects against anthracnose pathogens by two specific epiphytic fungi, En10 and En12 (Fig. 1). Subsequent identification revealed that En10 and En12 were classified as Aspergillus niger and Talaromyces, respectively (Fig. 2). This represents the first documented instance of their antagonistic activity against tea plant anthracnose.

      Aspergillus niger is a common species of Aspergillus fungi, which is widely found in food, plant products, and soil species[27]. It has a strong ability to produce enzymes, and can produce a variety of high-activity extracellular enzymes, such as cellulase, pectinase, amylase, acid protease, glucose oxidase, and so on. Therefore, it is widely used in the fermentation industry. Some studies have shown that adding Aspergillus niger during tea fermentation can promote the decomposition of tea protein and affect the quality of tea[28,29]. In this study, it was found that Aspergillus niger En10 had a significant inhibi tory effect on the growth of anthracnose pathogen colonies (Figs 1 & 3). After spraying 104/ml spore suspension of En10 on healthy tea leaves (Fig. 4), the incidence of anthracnose inoculation was significantly reduced. This suggests that Aspergillus niger En10 has potential as a biocontrol agent against tea anthracnose.

      Talaromyces are important decomposers in nature and some Talaromyces fungi can produce important enzymes. For example, Talaromyces verruculosus, Talaromyces pinophilus and Talaromyces purpureogenus can produce large amounts of cellulase and xylanase[30−33]. Some metabolites of Talaromyces can also enhance the absorption of minerals by plants, thereby improving the disease resistance and stress resistance of plants. A strain of Talaromyces En12 was identified, which could significantly improve the resistance of tea leaves to anthracnose after spraying a spore suspension (Fig. 4). Furthermore, the antagonistic activity of En12 may be attributed to its enhancement of the antioxidant capability of tea leaves, such as the activities of SOD and POD (Fig. 5). This finding highlights the potential of using epiphytic fungi, such as Aspergillus niger and Talaromyces, to be developed as low-cost biocontrol agents for effectively controlling tea plant anthracnose.

      Studies have shown that endophytes can improve plant resistance to pathogens through different mechanisms, such as producing secondary metabolites to inhibit the growth of pathogens and inducing plant defense mechanisms. Endophytes can also induce the production of polyphenol oxidase, peroxidase, phenylalanine ammoniase, and superoxide dismutase in plants to improve the disease resistance of host plants. Prabhukarthikeyan et al. found that after treatment by Pseudomonas fluorescens, the enzyme activities of POD, PPO, PAL, SOD and CAT in roots and stalks of turmeric have been significantly improved, and they have effectively prevented and controlled the root rot disease of turmeric[34]. Khan et al. isolated from the roots of tomato a penicillium strain LK5[35]. Its secondary metabolites can increase the activity of POD, CAT, PPO, and GSH, to improve the defense capability of the plant itself. In our investigation, significant alterations in SOD and POD enzyme activities in tea leaves were observed following treatment with En12 (Fig. 5). These changes potentially contribute to enhancing the tea plant's resistance against anthracnose pathogen incursion. Nonetheless, our primary focus centered on the screening and characterization of epiphytic fungi with efficacy against tea anthracnose. Further research is warranted to elucidate the mechanisms of epiphytic fungi action, isolate and identify antibacterial constituents, and explore the interactions between epiphytic fungi and their host tea plant. Overall, our findings demonstrate the potential of using epiphytic fungi as a cost-effective and environmentally friendly approach for controlling tea plant anthracnose.

    Conclusions

    • Thirteen strains of epiphytic fungi were isolated and purified from healthy tea leaves, with nine found to be non-pathogenic. Through plate confrontation tests, two strains, En10 identified as Aspergillus niger, and En12 identified as Talaromyces, were observed to significantly inhibit the growth of pathogens. Dual culture experiments and disease resistance trials on tea leaves demonstrated that En10 and En12 effectively suppressed the invasion of anthracnose disease. Additionally, En12 displayed potential to improve the antioxidant response of tea leaves, thereby increasing resistance to anthracnose. In all, this study identified two epiphytic fungi, belonging to Aspergillus niger and Talaromyces, that have the potential to be used as biological control agents for the management of tea plant anthracnose.

    Author contributions

    • The authors confirm contribution to the paper as follows: study conception and design: Wang W, Yu Y, Bao L; tea samples collection: Liu L, Jiao M, Ma Z, Ma Y, Zhou J; experiment conduction: Guan H, Liu L, Xie X, Bao Y; data analysis, visualization: Guan H, Liu L, Wang W; original draft preparation: Wang W, Liu L. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    • All data supporting the conclusions of this study may be found in the publication, which are available online. Any additional relevant information can be obtained from the corresponding author upon request.

      Acknowledgments

      • This work was supported by the National Natural Science Foundation of China (32202551), China Agriculture Research System of MOF and MARA (CARS-19) and the National key research and development program of China (2022YFD1602003). Some texts in this paper were polished by Stork's Writing Assistant (www.storkapp.me/writeassistant).

      Conflict of interest

      • The authors declare that they have no conflict of interest.

      • # Authors contributed equally: Lu Liu, Haonan Guan

      Rights and permissions
      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (5)  Table (1) References (35)
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    Liu L, Guan H, Jiao M, Ma Z, Bao Y, et al. 2024. Identification of two antagonistic fungi and antifungal activity analysis against anthracnose in tea plant (Camellia sinensis). Beverage Plant Research 4: e032 doi: 10.48130/bpr-0024-0020
    Liu L, Guan H, Jiao M, Ma Z, Bao Y, et al. 2024. Identification of two antagonistic fungi and antifungal activity analysis against anthracnose in tea plant (Camellia sinensis). Beverage Plant Research 4: e032 doi: 10.48130/bpr-0024-0020
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  • Introduction
  • Materials and methods

  • Plant material and reagents

  • Pathogenic fungi

  • Purification and morphological observation of antagonistic fungus

  • Molecular identification of antagonistic fungi and phylogenetic construction

  • Antifungal activity analysis

  • Dual culture tests
  • Disease resistance experiments on tea leaves

  • The determination of antioxidant enzymes' activity

  • Statistical analysis

  • Results

  • Isolation of epiphytic fungi and their antagonistic activity against the anthracnose pathogen

  • Inhibition of colony growth of anthracnose pathogen by antagonistic fungi

  • Control effect of antagonistic fungi against anthracnose on tea leaf

  • Activities of antioxidant enzymes after En10 and En12 treatment

  • Discussion
  • Conclusions
  • Author contributions
  • Data availability
  • Acknowledgments
  • Conflict of interest
  • Rights and permissions
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