ε-poly-L-lysine – Imidazole Ketone Erastin

Control eﬃciency and expressions of resistance genes in tomato plants treated with ε-poly-L-lysine against Botrytis cinerea

A B S T R A C T
The antifungal properties and the induction of resistance by ε-poly-L-lysine (ε-PL) were examined to reveal its potential in protecting tomato plants against Botrytis cinerea. As presented herein, ε-PL at 1200 mg/L was found to have optimal in vitro antifungal activities, achieving an inhibition rate of 94.96%. In ﬁrst-year ﬁeld tests, ε-PL (1200 mg/L) had a control eﬀect of up to 79.07% against tomato grey mould. Similar results were obtained in the second year. In greenhouse experiments, ε-PL was observed to eﬀectively reduce leaf infection, with an observed control rate at 89.22%. To deﬁne the molecular-genetic mechanisms, we compared the gene expression under four diﬀerent conditions: sterile water sprayed plants (Control), Botrytis-infected plants (Inf), ε-PL-treated plants (ε-PL) and ε-PL-treated + infected plants (ε-PL + Inf). Quantitative PCR analysis at 36 h after inoculation revealed that ε-PL + Inf plants exhibited signiﬁcant expression and priming of several key Botrytis-induced genes in tomato. The results indicate that ε-PL promoted plant capacity of tomato to activate defense mechanisms upon pathogen attack. In total, these ﬁndings revealed that ε-PL should be an excellent biocontrol agent candidate that combined direct antifungal activity against B. cinerea and plant resistance capacity.

1.Introduction
Botrytis cinerea, the causal agent of grey mould, causes large eco- nomic losses over the world, including on a variety of soft fruits, ve- getables and ﬂowers, and especially greenhouse-grown tomatoes (Lycopersicon esculentum). The pathogen often infects leaves, petioles, stems and fruits. B. cinerea has diﬀerent infection mechanisms with various host plants [1,2]. In general, the conidial germ tubes can not only penetrate through wounds or natural openings, but also directly penetrate healthy plant tissues, which result in the tissues being sec- ondarily invaded by mycelia from previous colonized, dead plant tis- sues [3].Alternative control methods of Botrytis diseases are urgently needed, as this pathogen is responsible for some of the most devastating diseases of plants across a wide range of climates. In support of this, the de- velopment of biocontrol agents as an alternative to fungicides has been explored as a means to control pathogen spread and disease [4]. Recent biocontrol research has shown that compared with the expression of the wild-type Streptomyces lydicus strain A01, the expression of Paenibacillus polymyxa β-1,3-1,4-glucanase gene in S. lydicus A01 highly improved its biocontrol eﬀect against B. cinerea [5]. Additionally, Kalogiannis et al.[6] found that the activity of Rhodotorula glutinis Y-44 protected tomato plants from grey mould infections, and its isolate performed as eﬀectively as fungicides in tomato plants. Combined with the use of commonly used fungicides, a recent report provided an alternative strategy that can be adopted in the place of, or be combined with, fungicides to enhance natural defense capabilities of plants by inducers [7].

The activation of defense signaling during Botrytis-plant interactions is widely studied, and in total, these processes are well-characterized as coincident with necrosis and the localized accumulation of de novo synthesized pathogenesis-related (PR) proteins. PR proteins are plant species-speciﬁc proteins produced in response to infection with viruses, fungi or bacteria. Some of these proteins particularly, chitinase and β- 1,3-glucanase, are hypothesized to provide defense against B. cinerea via hydrolytic action on its cell walls [5,8]. Further, the accumulation of various mRNA encoding PR proteins is also detected in leaves in re- sponse to the causal agents of powdery and downy mildews [9]. To date, however, the function of chemical inducers, such as ε-poly-L-ly- sine, has not been widely investigated.ε-poly-L-lysine (ε-PL), consisting of 25–30 L-lysine residues [10], is a homo-poly-amino acid characterized by a peptide bond between α- carboXyl and ε-amino groups. Yamanaka et al. [11] isolated an ε-PL- synthesizing enzyme, cloned its gene and then characterized the acti- vation and polymerization of lysine. ε-PL was ﬁrst discovered as one of Dragendorﬀ-positive compounds in the culture ﬁltrate of the actinomycete of Streptomyces albulus 346 isolated from the soil [12]. Recently, several ε-PL-producing strains were isolated, such as Strep- tomyces sp. M-Z18 [13], S. griseofuscus [14], S. aureofaciens [15], Ba- cillus subtilis [16] and Kitasatospora sp. PL6-3 [17]. Because of its ex- cellent biodegradability, heat stability, antimicrobial activity and low toXicity [18], ε-PL is widely used as a food additive to extend the shelf-life of meat, rice, cooked vegetables and other food products in Japan, Korea, the United States as well as other countries [19]. Historically, ε- PL has been used for medical research, in large part based on its unique biologic activities, including the ability to reduce cell toXicity by Tomato seeds (L. esculentum, Oulong) were obtained from the College of Plant Protection, Northwest A & F University, Shaanxi, China. Two experiments with three treatments, ε-PL (1200 mg/L), procymidone (positive control), and tap water (negative control), were carried out for two consecutive years from January 2015 to May 2016 in the ﬁeld (Yangling, China). In ﬁeld applications, the same method was used in the ﬁrst year and the second year. The experiment adopted a random block design with four replicates.

The day and night tempancreatic lipase activity and the production of oral bacterial toXin [20]. Interestingly, the microbicidal activities of ε-PL depend on mi- crobial cell sizes rather than the microbial species [21]; recent support for this was provided by Bo et al. [22], who reported that ε-PL treat- ment resulted in increased levels of ergosterol and fatty acids in cell membranes of Saccharomyces cerevisiae. Meanwhile, ε-PL also induces osmotic stress by breaking ionic balance between inner and outer membrane of the fungus. Yamanaka and Hamano [20] suggested that the antimicrobial mechanism of ε-PL was based on the electrostatic interaction with the microbial cell surface, which results in membrane disorganization, abnormal cytoplasm distribution, and the physiolo- gical damage of microbial cells. Because of its broad ranges of anti-microbial activities (including Gram-negative and Gram-positive bac- teria, yeasts and molds) [15,23], ε-PL is widely employed to inhibit some pathogens. However, there have been very few studies conducted on inducing resistance to B. cinerea in tomato plants. Herein, we de- scribe the antifungal activity of ε-PL against B. cinerea and the eﬀects of ε-PL in defensive responses of tomato plants by using real-time quan- titative PCR (RT-qPCR).

2.Materials and methods
2.1.Fungal isolation and storage
B. cinerea strain B05.10 was provided by the Laboratory of Plant Pathology, College of Plant Protection, Huazhong Agricultural University, Wuhan, China. B. cinerea was cultured on potato dextrose agar (PDA) medium.

2.2.Preparation of spore suspension
B. cinerea spore suspensions were prepared using spores collected from a one-week pathogen culture, suspended in sterile water supple- mented with 0.3% Tween-80. The spore suspension was ﬁltered to adjust the concentration of the suspension to 1.0 × 106 conidia/mL using a sterile sieve. The conidial spore concentration was measured using Bürker chamber.

2.3.Eﬀect of ε-PL on the mycelial growth of B. cinerea in solid media
The antifungal activity of ε-PL was assessed by the radial growth test on PDA. The ε-PL solutions of eight diﬀerent concentrations were pre- pared with sterile water supplemented with 0.3% Tween 80. ε-PL so- lutions were miXed with sterile PDA to prepare PDA mediums con-
taining 1 × 102, 2 × 102, 4 × 102, 6 × 102, 8 × 102, 1 × 103, 1.2 × 103, and 1.4 × 103 mg/L. PDA solutions were poured into 90 mm Petri dishes, and 5 mm plugs of B. cinerea collected in the outer areas of active cultures were inoculated onto the prepared plates. Three replicates in each treatment were evaluated. Procymidone (80% Wettable powder, WP, Shaanxi Meibang Agrochemical Co., Ltd.) was used as the positive control. On the 4th day after incubation with a regular photoperiod of 12 h light/12 h dark at 23 °C, the diameters of the colonies were measured to calculate the inhibition rates and 50% eﬀective concentration (EC50) [24]. The experiment was replicated three times. 14–18 °C and 70%–85%. Each treatment was composed of three 90 m2 (6 m × 15 m) plots. Oulong, a susceptible tomato line, was planted with the row and plant spaces of 40 cm and with plastic ﬁlm mulched. Tomato leaves were sprayed evenly with diﬀerent test agents and spraying quantity was 18 L per 90 m2 tomato plants. All the sprayings were done at the same time.

Disease development was recorded on the 7th day after each ap- plication. The disease severities of leaves were scored using the fol- lowing scale: 0 = no diseased leaves; 1 = 0–5% of leaves covered with lesions; 3 = leaves with infection lesions making up 6–10%; 5 = leaves with infection lesions making up 11%–20%; 7 = leaves with infection lesions making up 21%–40%; and 9 = leaves with infection lesions making up 41%–100%. The disease index on leaf was calculated with the following equations:Disease index (DI) = [Σ (the number of diseased plant leaves at a disease score × the disease score) / (total plant leaves in- vestigated × 9)] × 100%.Following the protocol enacted by the Bioassay Lab, Institute for the Control of Agrochemicals, Ministry of Agriculture, China (1993), the control eﬀects were calculated by the following formula:E =[(C − T)/C] × 100. Where E is the control eﬀect (%); C is the DIs of tap water to the control plot; and T is the DIs of chemical application in the test plot.

2.5.ε-PL induction to resistance in the pot experiments
Seeds were surface sterilized with 3% sodium hypochlorite for 3 min and immediately rinsed with sterile distilled water three times. Each seed was planted in 15 cm diameter pot ﬁlled with sterile soil in the greenhouse. At the 5–6 leaf stage, ε-PL (1200 mg/L) was sprayed onto the leaves and B. cinerea B05.10 mycelium discs (0.5 cm diameter) were inoculated onto leaves at intervals of 1, 3, 5, and 7 days after ε-PL spraying. Each treatment consisted of three replicates, with siX potted plants sprayed with 40 mL ε-PL each replicate. The control plants were sprayed with distilled water. The greenhouse temperature was kept at 23 °C, and the relative humidity was between 90% and 95%. The size of lesions was measured on the 3rd day after inoculation [25]. The ex- periments were repeated three times.

2.6.RNA isolation and RT-qPCR assays
Tomato leaves on pot experiments were used for RT-qPCR assays. ε- PL treatment was performed 3 days prior to B. cinerea spores or water inoculation. In total, we analyzed four conditions: untreated and sterile water sprayed (Control), untreated and Botrytis-infected plants (Inf), ε- PL-treated and non-infected plants (ε-PL), and ε-PL-treated and infected plants (ε-PL + Inf). For gene expression analysis, leaves treated with the chemical were separately collected 0, 12, 24, 36, 48, 72 and 96 h after inoculation and stored at −80 °C until processing. Total RNA was prepared using BIOZOL total RNA extraction kit (Bioer, China) fol- lowing the manufacturer’s instructions. RNA integrity was determined by gel electrophoresis, and the RNA amount was calculated using a Qubit 2.0 Fluorometer (Life Technologies, USA). Total RNA (1 μg) was used for ﬁrst strand cDNA synthesis using the single strand cDNA synthesis kit (Gene Copopeia, USA) with oligo (dT)18 primer (MBI Colony diameters were measured 4 days after inoculation. In the table, values are mean ± SE. In the same columns, diﬀerent superscript letters indicate signiﬁcant dif- ferences at P < 0.05 by Duncan's test Fermentas, Vilnius, Lithuania).The IQ™5 Real-Time PCR System (BioRad, California, USA) was employed for quantitative PCR ampliﬁcations. The GAPDH gene was used as the internal constitutively expressed control in the RT-qPCR analysis [26]. The primers used in RT-qPCR for genes expression patterns analysis are listed in Table 1. For the real-time PCR reaction, 1 μL total cDNA was added to a 20 μL PCR reaction miXture containing 10 μL of 2 × ultra SYBR miXture (CWBIO, Beijing, China), 0.4 μL of each primer, 2 μL template, and 7.2 μL of water. Real-time PCR conditions were as follows: denaturing at 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C, 30 s at 56.5 °C, 30 s at 72 °C. Each reaction included a non-template control. All analyses were performed in biological tripli- cate. Relative transcript quantiﬁcation was calculated by the com- parative 2−ΔΔCT method [27]. 2.7.Data processing Statistical analyses were performed using SAS (ver. 9.2). All com- parisons at speciﬁc times were carried out using the Student's t-test at In the table, values are mean ± SE. In the same columns, diﬀerent superscript letters indicate signiﬁcant diﬀerences at P < 0.05 by Duncan's test.P < 0.05. 3.Results 3.1.Inhibitory eﬀect of ε-PL on mycelia growth of B. cinerea The mycelial growth of B. cinerea was inhibited by increasing the concentration of ε-PL from 100 to 1400 mg/L (Table 2). Four days after inoculation with B. cinerea, ε-PL signiﬁcantly inhibited mycelial growth (P < 0.05) at all the diﬀerent concentrations but 100 mg/L. The in- hibition ratios of ε-PL (400 mg/L) were stronger than those with pro- cymidone. Compared with the control, ε-PL separately obviously in- hibited the mycelial growth at 1400 and 1200 mg/L by 100% and 94.96%, but its inhibitions at the two concentrations did not diﬀered signiﬁcantly (P > 0.05). In addition, the 50% eﬀective concentration (EC50) of ε-PL was 271.83 mg/L. The regression equation was:
y = 2.8235x – 1.8732 with an R2 value of 0.8510.

3.2.Ability of ε-PL to reduce grey mould in the ﬁeld
ε-PL (1200 mg/L) and procymidone signiﬁcantly reduced tomato grey mould in the ﬁeld. During the ﬁrst-year ﬁeld trial, application of ε- PL signiﬁcantly decreased DI by compared with sterile water controls The concentration of ε-PL adopted was 1200 mg/L. Procymidone was prepared by adding 1 g 80% procymidone to 1000 mL water.In the table, values are mean ± SE. In the same columns, diﬀerent superscript letters indicate signiﬁcant diﬀerences at P < 0.05 by Duncan's test. Fig. 1. Relative transcript levels of β-1, 3-glucanase related genes to B. cinerea in tomato leaves by quantitative real-time PCR. Error bars are standard deviation from three independent experiments. Diﬀerent superscript letters indicate signiﬁcant diﬀerences at P < 0.05 by Duncan's test.A: only sterile water sprayed (Control); B: only B. cinerea spores inoculated;C: only ε-PL sprayed; D: ε-PL treatment was performed 3 days prior to B. cinerea spores inoculation(Table 3). Interestingly, the DI increased slightly more with ε-PL ap- plication than with procymidone. These data suggested that ε-PL and procymidone had the signiﬁcant control eﬃciencies of 79.07% and 86.72% (P < 0.05), and there was no signiﬁcant diﬀerence between ε- PL application and procymidone. During the second year, ε-PL and procymidone had the signiﬁcant control eﬃciencies of 73.61% and77.27% to reduce grey mould in the ﬁeld (P < 0.05). 3.3.ε-PL induces resistance in tomato plants On day 1, 3, 5, and 7 after spraying plants with ε-PL, B. cinerea was inoculated onto the leaves of tomato. As shown in Table 4, ε-PL was observed to perform better on the 3rd day after spraying than that observed for days 1, 5 and 7. The control eﬃciency of ε-PL (1200 mg/L) was determined on the 3rd day after inoculation. On the 3rd day after spraying ε-PL onto plants, the lesion sizes on tomato plants were de- creased compared with the control plants, and the control eﬃciency was quantiﬁed at approXimately 89%. On the 5th and 7th day after spraying ε-PL, the control eﬃciencies were found to gradually decrease by 55.56% and 22.40%, respectively. 3.4.Transcript level of Glucanase A and Glucanase B Glucanase A and Glucanase B are two important genes associated with β-1,3-glucanase. Both genes were signiﬁcantly expressed in ε-PL + Inf plants (P < 0.05). Glucanase A expression was sharply increased at 24 h after inoculation and peaked in Inf plants (Fig. 1). The expres- sion of Glucanase A at 24 h after inoculation was 8.83 times that in control plants. In ε-PL plants, Glucanase A expression at 12 h after spraying was up-regulated and 1.46 times that the control group, and the expressions of Glucanase A at the other time points was reduced. In ε-PL + Inf plants, the transcription of Glucanase A peaked at 36 h after inoculation, with an observed level of expression > 7 times that ob- served in control group.

Fig. 2. Relative transcript levels of genes related chitinase to B. cinerea in tomato leaves by quantitative real-time PCR. Error bars are standard deviation from three independent experiments. Diﬀerent superscript letters indicate signiﬁcant diﬀerences at P < 0.05 by Duncan's test.A: only sterile water sprayed (Control); B: only B. cinerea spores inoculated;C: only ε-PL sprayed;D: ε-PL treatment was performed 3 days prior to B. cinerea spores inoculation.Similarly, the expression of Glucanase B at all the time points was observed to be up-regulated in Inf plants in comparison with the ex- pression in control plants. The expression of Glucanase B increased since 12 h after inoculation, and peaked at 36 h after inoculation, being ~ 12 times that in control group. In ε-PL plants, the expression of Glucanase B at 48 h after spraying was signiﬁcantly increased to the highest level being 45.40 times that in control group (P < 0.05). In ε-PL + Inf plants, the expression of Glucanase B was slightly increased at 12 and 24 h after inoculation. The transcription of Glucanase B peaked at 36 h after inoculation, with an approXimate 55-fold increase compared with the control expression. This indicated that Glucanase B could play an important role in the process of defense response triggered by pathogen infection. 3.5.Transcript level of Chitinase 3 and Chitinase 9 Chitinase 3 and Chitinase 9 are two important genes associated chitinase. Chitinase 3 and Chitinase 9 expression levels were sig- niﬁcantly stimulated in ε-PL + Inf plants (P < 0.05). Compared with the control expression, the relative expression levels of the two genes at each other collection time points were shown in Fig. 2. In Inf plants, Chitinase 3 expression at 48 h after inoculation was ~ 17 times that in control group, and its expression at the other time points were all down- regulated. At the 12, 48 and 96 h after spraying in ε-PL plants, the expressions of Chitinase 3 tend to be slightly up-regulated to be 1.90, 2.25, and 1.75 times that in control plants, respectively. Likewise, the expression of Chitinase 3 was slightly up-regulated since 24 h after inoculation in ε-PL + Inf plants and peaked at the 36 h after inoculation, and the peak was 42.96 times that in control group.Chitinase 9 expression was up-regulated to diﬀerent levels at all the time points in Inf plants. The expression of Chitinase 9 at 48 h after inoculation was the highest peak, 5.61 times that in control group. In ε- PL plants, the expression of Chitinase 9 at 12 h after spraying with a peak was 13.02 times that in control group. In ε-PL + Inf plants, the expression of Chitinase 9 was slightly up-regulated at 24 h after in- oculation, and peaked at 36 h after inoculation, being 25.29 times that in control group. Therefore, Chitinase 3 and Chitinase 9 could be related to defense response triggered by pathogen infection. 3.6.Transcript level of PR1b1 ε-PL can induce resistance of tomato plants to B. cinerea by in- creasing expression of a pathogenesis-related protein gene. At all the time points except the 12 and 24 h in Inf plants, the expression of PR1b1 was all signiﬁcantly up-regulated compared with PR1b1 expression in control group (P < 0.05), and PR1b1 gene presented an obvious peak at the 48 h after inoculation, which was 6.41 times that in control group. In ε-PL plants, the expression of PR1b1 at 96 h after spraying presented the highest peak, being 4.40 times that in control group (Fig. 3). In ε-PL + Inf plants, the expression of PR1b1 appeared up- regulated at the 24 and 36 h after inoculation, and expressions of PR1b1 were 1.80 and 8.06 times that in control group, respectively, but pre- sented down-regulated expressions at the other time points. It followed that PR1b1 could be involved in resisting tomato grey mould. 4.Discussion The antifungal activities of ε-PL have been previously demonstrated to reduce mycelial growth of many fungi in vitro [28–30]. However, its impact on B. cinerea, the causal agent of tomato grey mould, remains largely undeﬁned. In the current study, the inhibition rate of ε-PL against B. cinerea was investigated, and as demonstrated, was observed to have a higher speciﬁc activity than similarly tested compounds [31,32]. ε-PL exhibits the characteristics of water solubility, biode- gradability, stability and low toXicity [33]. In this study, our results demonstrated that ε-PL could eﬀectively control tomato grey mould caused by B. cinerea. Herein, we demonstrate that ε-PL progressively reduced mycelial growth of B. cinerea. At 1200 and 1400 mg/L, ε-PL resulted in strong antifungal activity on B. cinerea, a process that we hypothesize is the result of the induction of damage to conidia plasma membrane, resulting in the loss of cytoplasmic materials from the hy- phae of B. cinerea [34,35].Unique to the current study, our ﬁndings highlight that ε-PL ap- plication against tomato grey mould can likely serve as a preventive measure in the ﬁeld, as supported by multiple years of ﬁeld-based ex- perimentation. Indeed, in both years of study, ε-PL and procymidone had signiﬁcant control eﬃciencies of the disease. These results are consistent with the report of Macnish et al. [36]. In addition, the control eﬃciency of ε-PL against B. cinerea was observed to yield a higher ef- ﬁcacy as compared to previously tested chemical compounds in similar ﬁeld studies [4]. While there are many environmental factors, such as temperature, humidity, pH and microorganisms, which consistently impact the eﬃcacy of ε-PL in the ﬁeld experiment, our data provide prima facie evidence that while further tests under varied environmental conditions are needed, the use of ε-PL to manage B. cinerea infection is a viable management option. Compared with lesion sizes on control plants, the lesion sizes of tomato plants decreased on the 3rd day after ε-PL was sprayed (1200 mg/L) (Table 4). The control percentages of ε-PL gradually decreased on the 5th and 7th day after spraying ε-PL. The results indicated that ε-PL could signiﬁcantly reduce tomato grey mould by inducing resistance on the 3rd day after spraying ε-PL. While the data presented herein are promising, as a function of the molecular-genetic signaling processes underpinning this activity it is evident that the antifungal mechanism(s) of ε-PL is complex, likely involving a combination of direct fungi-toXic property and the elicitation of host-speciﬁc responses [37]. Thus, further studies on ε-PL are necessary to fully elucidate the defense-associated signaling processes. The results presented herein further demonstrate that the regulation of speciﬁc defense-associated transcripts was quickly up-regulated at 36 h after inoculation in ε-PL + Inf plants. This result illuminates that tomato can receive the stimuli from B. cinerea quickly and trigger β-1,3- glucanase, chitinase, and other PR proteins to protect themselves from being invaded; moreover, these data are in agreement with Puthoﬀ et al. [38], who reported that Glucanase B, Chitinase 9 and PR-1 tran- scripts become abundant in the whiteﬂy-infested leaves at day 5 after infestation, and Chitinase 3 also increases although less abundant. In this study, ε-PL treatment has shown to up-regulate the gene expression of β-1,3-glucanase and chitinase. Therefore, ε-PL might be beneﬁcial for tomato plants against B. cinerea by inducing β-1,3-glucanase and chit- inase activities. β-1,3-glucanase has been shown to have an indirect eﬀect on plant defense by causing the formation of oligosaccharide elicitors, which elicit the production of other PR-proteins or low mo- lecular weight antifungal compounds, such as phytoalexins [39–41].There are several other reports on the induction of β-1,3-glucanases along with chitinases and many other PR-proteins during pathogen infection [42–45]. In this study, the expressions of Chitinase 3 and Chitinase 9 peaked at 48 h after inoculation in Inf plants. However, Benito et al. [46] reported that the expressions of Chitinase 3 and Chitinase 9 at 20 °C peak at 72 and 32 h after inoculation, respectively. This ﬁnding is in agreement with the observations of Sridevi et al. [47] on the role of chitinase in resistance of rice plants to Rhizoctonia solani. Similarly, in tomato, investigations of Chen et al. [8] revealed the in- volvement of chitinase in defense ε-poly-L-lysine of the plants against B. cinerea as a destructive phytopathogen with necrotrophic lifestyle. In addition, ε-PL can induce the resistance of tomato leaves more strongly in ε-PL + Inf plants than in Inf plants. This phenomenon is likely attributed to either accumulated resistance signaling or the production of new resistance- inducing eﬀectors.