Biocontrol of strawberry Botrytis gray mold and prolong the fruit shelf-life by fumigant Trichoderma spp.

doi:10.21203/rs.3.rs-2868489/v1

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Research Article

Biocontrol of strawberry Botrytis gray mold and prolong the fruit shelf-life by fumigant Trichoderma spp.

https://doi.org/10.21203/rs.3.rs-2868489/v1

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Objectives

To screen high active VOCs-producing Trichoderma isolates against strawberry gray mold caused by Botrytis cinerea, and to explore their antagonistic mode of action against the pathogen.

Results

VOCs produced by nine Trichoderma isolates (Trichoderma atroviride T1 and T3, Trichoderma harzianum T2, T4 and T5, T6, T7, T8 and T9 identified as Trichoderma asperellum by ITS sequencing in this work) significantly inhibited the mycelial growth (13.9 − 63.0% reduction) and conidial germination (17.6 − 96.3% reduction) of B. cinerea, the highest inhibition percentage belonged to VOCs of T7; in a closed space, VOCs of T7 shared 76.9% and 100% biocontrol efficacy against gray mold on strawberry fruits and detached leaves, respectively, prolonged the fruit shelf-life by 3 days in presence of B. cinerea, completely protected the leaves from the pathogen infection; volatile metabolites of T7 damaged the cell membrane permeability and integrity of B. cinerea both on water agar or on strawberry leaf surface, thereby inhibiting the mycelia growth, conidia germination and the formation of infection pads.

Conclusions

T. asperellum T7 showed high biofumigant activity against mycelial growth especially conidial germination of B. cinerea and thus protected strawberry fruits and leaves from gray mold, which acted by damaging the pathogen’s plasma membrane and resulting in cytoplasm leakage, was a potential biofumigant for controlling pre- and post-harvest strawberry gray mold.

Antagonism

Botrytis cinerea

Fragaria × ananassa

Trichoderma

Volatile organic compounds

Strawberry (Fragaria × ananassa Duch.) is an economically important crop in China, however, its fruits are highly perishable due to their extreme tenderness, vulnerability to mechanical damage, high level of respiration and their susceptibility to fungal spoilage, and responsible for 20 − 30% postharvest losses for strawberry industry (Dennis 1978; Guo 2016). Among them, gray mold caused by necrotrophic fungus Botrytis cinerea is one of the most destructive diseases worldwide (Romanazzi et al. 2012). In strawberries, serious losses caused by the pathogen can be traced to infections that occur either between flowering and fruit maturity or during harvesting and subsequent handling, storage, marketing and even after purchase by the consumer. Nearly all parts including fruits, flowers, leaves, petioles and crown can be infected. Under low temperature, high humidity and poor ventilation conditions, yield loss can exceed 50% (Sutton 1998). Fruit infection often begins during blossoming when dispersed conidia arrive on flower parts, where they cause quiescent infections that only express disease once the fruit matures (Powelson 1960). When fruits develop the characteristic gray mold, that spreads rapidly and causes fruit rot due to cell-wall-degrading enzymes and phytotoxic metabolites such as botrydial secreted by the pathogen (Qin et al. 2017). Thus far, developing strawberry varieties with resistance to gray mold disease is challenging due to the high susceptibility of most genetic types and the difficulty in determining the source of natural genetic resistance (Zhang et al. 2019; Caseys et al. 2021; Petrasch et al. 2022). Therefore, intensive efforts have been made to minimize these losses through chemical, physical, biological and nanotechnological measures, individually or combinedly (Li et al. 2014). Application of fungicides has been considered as the most efficient way to control B. cinerea. However, in China, the efficacy of some fungicides decreased because of the pathogen having developed resistance to the commonly used fungicides including sprocymidone, carbendazim, diethofencarb and fludioxonil (Ji 2021), and some others have withdrawn from the market because of toxicological problems (Bi et al. 2017).

A viable alternative to protect pre- or post-harvest fruits and vegetables from fungal decay is use of biological control agents (BCAs) (Calvo et al. 2010; Navarta et al. 2014; Guo et al. 2019; Esparza-Reynoso et al. 2022), such as fungi, bacteria, and actinomycetes have shown great potential to control fruits decay, mainly though several mechanisms, including competition for nutrients and space, parasitism, lytic enzymes, induced resistance, and antimicrobial substances such as non-volatile and volatile organic compounds (VOCs) (Wang et al. 2010; Wang et al. 2013; Di Francesco et al. 2015; Wei et al. 2016; Oregel-Zamudio et al. 2017; Qin et al. 2017; Oro et al. 2018; Wang et al. 2018; Boukaew et al. 2019; Guo et al. 2019). VOCs are small molecules emitted by a huge variety of organisms, which can not only influence the architecture and growth of plants (Hung et al. 2013; Kottb et al. 2015; Lee et al. 2016), but also enhance plant defense (Kottb et al. 2015; Di Francesco et al. 2016; Werner et al. 2016). Among the VOCs-producing fungi, Trichoderma is one of the best-studied beneficial fungi (Esparza-Reynoso et al. 2022). VOCs of Trichoderma asperellum (strain IsmT5) induce phytohormones such as salicylic acid (SA) or abscisic acid (ABA) in leaves, H₂O₂ or substances related to pathogen attack like camalexin and phytoalexins, and genes related to defense and stress such as pathogenesis-related protein 1 (PR1) accumulating in plants cocultivated with T. asperellum (strain IsmT5), which enhance both growth-manipulating function and the expression of defense-related genes and the resistance to B. cinerea (Kottb et al. 2015).

However, according to China Pesticide Information Network, the BCAs registered for controlling strawberry gray mold are Bacillus species, such as Bacillus subtilis (PD20151456, Shandong Zhongnuo Pharmaceutical Co., Ltd.), and Bacillus amyloliquefaciens QST713 (PD20211364, Bayer Co., Ltd.) (http://www.chinapesticide.org.cn/), and so on. Due to the increasing demand for chemical-free crop products globally, the main objectives of this work aimed to screen high active VOCs-producing Trichoderma isolates against strawberry gray mold caused by B. cinerea, and to explore their antagonistic mode of action against the pathogen, in terms of the production of VOCs.

Fungal strains and growth conditions

For this study, nine isolates of Trichoderma numbered as T1, T2, T3, T4, T5, T6, T7, T8, and T9, respectively were used. Among them, T. atroviride T1 (OP009872), T. harzianum T2 (OP010005), T. atroviride T3 (OP010004), T. harzianum T4 (OP010007) and T. harzianum T5 (OP019728) have been isolated and identified in a previous research (Hu et al. 2022). The remaining four strains, T6 to T9 were isolated from peanut rhizosphere soil in Qingdao, Shandong Province, and have yet to be identified by morphological characteristics and ITS sequencing.

Botrytis cinerea was isolated from rotten fruits of strawberry and identified by morphological and molecular methods. Molecular identification was performed using ITS1/ITS4 primers, and sequencing results confirmed the isolated strains as B. cinerea (OQ789962).

All the isolated fungal strains were stored in a 20% (v/v) glycerol solution at -80°C in the Department of Plant Pathophysiology, College of Plant Health and Medicine, Qingdao Agricultural University, China.

In this work, B. cinerea was incubated on potato dextrose agar (PDA) at 25˚C for 7 d to collect conidia. The conidia were suspended with sterile distilled water containing 0.1% v/v Tween 80 and then filtered through four layers of sterile cheesecloth (Card et al. 2009). Concentrations of the conidia were determined using a hemocytometer and adapted to 1×10⁵ conidia/mL for the inoculation.

Molecular identification of Trichoderma isolates

Four strains of Trichoderma T6 to T9 were identified by internal transcribed spacer (ITS) sequencing. The genomic DNA of selected strains was extracted by cetyltrimethylammonium bromide (CTAB) lysis. Then using the universal primer pair ITS1(5'-TCCGTAGGTGAACCTGCGG-3')and ITS4༈5'-TCCTCCGCTTATTGATATGC-3'༉to amplify the ITS sequence of the extracted DNA (White et al. 1990). Using the thermal cycler to construct a PCR reaction system of the 20 uL reaction mixture containing the following: 10 µL 2X accurate taq master mix, 1 µL of 20 µM forward primer, 1 µL of 20 µM reverse primer, 1 µL DNA template, and 7 µL nuclease-free water. Amplifications were performed under the following conditions: initial denaturation at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 30 s, 59°C for 30 s, 72°C for the 60 s, and the final extension at 72°C for 10 min followed by 40 min cooling at 4°C. Amplification products were tested in 2% agarose gel and then submitted to Beijing Tsingke Biology Co., Ltd. (Qingdao, China) for sequencing, and the sequence data were aligned using the NCBI database (https://www.ncbi.nlm.nih.gov/).

The phylogenetic evolutionary tree was constructed by the neighbor-joining method using MEGA11, and the phylogenetic tree was examined by Bootstrap with 1000 replications. During phylogenetic analysis, Nectria cinnabarina (HM484690) was served as the out-group taxon (Mariela et al. 2017).

In vitro antagonistic activity of VOCs of Trichoderma isolates against B. cinerea

Inhibition of mycelial growth of B. cinerea

Antagonistic capability of the nine Trichoderma isolates to B. cinerea was determined by constructing a double Petri dish assay method (Qin et al. 2017). A 5-mm mycelial disc of Trichoderma isolates were individually pre-inoculated on PDA plates (9 cm in diameter) at 25 ℃ for 48 h. The plate lids were removed, the base PDA plate inoculated with B. cinerea was placed over the plate inoculated with Trichoderma isolates, then firmly sealed with parafilm to prevent any loss of VOCs and incubated at 25°C. A blank PDA plate was used instead of a Trichoderma plate as control. When the colonies on the control plate just reached the edge of the plate, the colony diameters (cm) of B. cinerea were recorded, and inhibition percentage was calculated as following formula: $I \left(\text{%}\right)=\left[\right(C-{T}_{n})/C]\times 100$, where C and T_n are the average colony radius (cm) of the pathogen in control treatment and in presence of antagonistic fungus, respectively (Živković et al. 2010).

Inhibition of conidial germination of B. cinerea

In the conidial germination experiment, a double Petri dish assay method was used (You et al. 2022). 100 µL of 1 × 10⁵ conidia/mL conidia suspension of B. cinerea were well coated on a plate, containing 2% sterilized water agar, then placed over the 2-day pre-inoculated PDA plates of Tricorderma isolates, respectively, sealed firmly with parafilm and incubated at 25 ℃ in darkness. The control plates contained B. cinerea without the antagonists. Twenty-four hours after inoculation, the conidial germination of B. cinerea was counted under an optical microscope LEICA DM750. For each treatment, 50 conidia were assessed and three replicates were performed. The inhibition percentage was calculated as the following formula: $I \left(\%\right)=\left[\right(Nc-Nt)/Nc]\times 100$, where Nc and Nt are the average germinated conidia number of the pathogen in control treatment and in presence of antagonistic fungus, respectively. A conidium was considered to have germinated if the length of the germ tube was larger or equal to the conidial diameter.

In the above experiments, VOCs produced by Trichoderma T7 had the best inhibition percentage against the pathogen, therefore, T7 was selected for the following studies.

Damage to the plasma membrane of B. cinerea caused by VOCs of Trichoderma T7

To assess the damage, a modified double-culture dish method described by You et al. (2022) was used. 50 µL of 1 × 10⁵ conidia/mL conidia suspension of B. cinerea was well distributed on a slide containing 2% sterilized water agar (WA), then the slide was attached in the plate lid with double-sided tape, and placed over the 2-day pre-inoculated PDA plates of Tricorderma T7, sealed firmly with parafilm. PDA plate without Trichoderma T7 was served as control. After incubation at 25 ℃ for 72 h, the slides were taken out and washed twice with 500 µL of 1 µg/mL propidium iodide [PI, in 0.01mol/L of pH7.4 phosphate buffer solution (PBS)], then stained with PI at room temperature for 30 min, washed twice with PBS, and immersed in PBS at room temperature for 10 min. All the experiments were carried out in the dark. The treated cells were observed under fluorescence microscope (BX53F, OLYMPUS) using excitation and emission wavelengths of 535 and 615 nm, respectively (Li and Zhu 2021).

Antagonistic activity of VOCs produced by T7 on gray mold on strawberry fruits

To evaluate the biocontrol efficacy, a modified method described by You et al. (2022) was used. Strawberry fruits (Fragaria × ananassa Duch, cv. Akihiime) were harvested from a strawberry greenhouse in Qingdao. Fruits with the same size and age were collected from healthy plants by hand at the commercial maturity stage in the morning and then transported to the laboratory, disinfected in 0.1% sodium hypochlorite for 2 min, washed twice in sterile distilled water, and air-dried on a worktable for 30 min, and then each fruit was wounded (1 mm × 1 mm × 1 mm) once with a glass capillary in the middle and then inoculated with 15 µL of 1 × 10⁵ conidia/mL the pathogenic spore suspension (in 0.5 mg/mL KH₂PO₄, containing 0.1% Tween 80 and 0.5 mg/mL glucose) in the wound, kept on worktable at room temperature for 30 min to promote the suspension to be absorbed. Bioassays were carried out in a sealed glass desiccator (diameter × height = 18 cm × 23 cm, a volume of about 5.8 L, disinfected in 75% ethanol under UV-C for 15 min). Three PDA plates inoculated with T7 (72-hour old) were placed at the bottom of the desiccator (B.c + T7), the treated strawberry fruits were placed on porcelain plates above the desiccator to keep enough distance from the bottom. Afterwards, The desiccators were quickly sealed with double-layer parafilm, incubated at 25 ℃ in 12 h light/darkness for 7 days. Three PDA plates uninoculated with the Trichoderma were served as positive control (B.c-only), strawberry fruits inoculated with three plates of T7 and 15 µL sterile water instead of B. cinerea were served as negative control (T7-only). Referring to the classification scale of Huang et al. (2011), strawberry fruits exhibiting the symptoms of gray mold in each desiccator were scored for disease severity using a scale of 0 to 8, where 0 represents healthy and 1, 2, 3, 4, 5, 6, 7 and 8 represent < 12.5, 12.6 to 25.0, 25.1 to 37.5, 37.6 to 50.0, 50.1 to 62.5, 62.6 to 75.0, 75.1 to 87.5 and 87.6 to 100% decay areas, respectively. The disease index was calculated for each treatment using the following formula:

$$Disease index=100\times \sum _{i=0}^{n}(Ln\times n)/8\times \sum _{i=0}^{n}\left(Ln\right)$$

where, n: the rating scale, Ln: the number of the fruit corresponding to the rating scale n.

The biocontrol efficacy of T7 against gray mold was calculated as the follows: $Be=\left[\right(DIc-DIt/DIc]\times 100$, where DIc and DIt are the average disease index of strawberry fruits inoculated with the pathogen in control treatment and in presence of antagonistic fungus, respectively.

Antagonistic activity of VOCs produced by T7 on gray mold on strawberry leaves

To evaluate the biocontrol efficacy of VOCs of T7 on gray mold occurred on strawberry leaves, detached leaves of the same size and age were sampled from healthy strawberry plants (F. × ananassa Duch, cv. Akihiime), immersed in 2% sodium hypochlorite solution for 3 min to make surface disinfection, followed by rinsing 3 times with sterile distilled water, dried and wrapped the petioles with sterilized absorbent cotton soaked in sterilized water. A modified inoculation method similar to fruits mentioned above was used (Gao et al. 2018), every leaf was wounded (0.5 mm in diameter) 3 times with a sterilized inoculation needle and then inoculated with the pathogenic spore suspension, kept on worktable at room temperature for 30 min, transferred into srerilized glass desiccators and sealed with two-layer parafilm, incubated at 25 ℃ in 12 h light/darkness. Eight days after inoculation, the disease index (DI) for the pathogen and the biocontrol efficacy of T7 to gray mold was calculated based on the above-mentioned formula. The leaves were sampled for staining and evaluating the antagonistic activity of VOCs from T7 against mycelial growth and conidial germination of the pathogen on leaf surface.

Antagonistic activity of VOCs from Trichoderma T7 against B. cinerea on strawberry leaves

The collected strawberry leaves were stained based on a modified method of Wees (2008). Place the leaves to be destained in a 50-mL Erlenmeyer, cover the leaves with 95% ethanol and heat in water bath at 90 ℃ for 30 min, then the leaves were transferred into a 50-mL Erlenmeyer and stained in trypan blue solution (mixing 2.5 mg/mL trypan blue in lactophenol with 95% ethanol in a ratio of 1 to 2 by volume) in water bath at 90 ℃ for 10 min, rinsed 3 times in sterile distilled water, then removed the solutions and covered the leaves in 70% glycerol. Mycelia and conidia of the pathogen on leaves were observed and microphotographed under microscope.

Statistical Analysis

All data were analyzed using one-way ANOVA at 0.05 level in SAS software (version 8.0), and graphs were generated using Origin software (version 2021). Three replicates were prepared for each isolate/treatment, all experiments were carried out twice, unless otherwise mentioned.

Molecular characteristics of Trichoderma isolates

BLAST results showed that the ITS-rDNA gene sequences of Trichoderma T6-T9 displayed high levels of similarity with Trichoderma asperellum (KT876621). Furthermore, phylogenetic analysis obtained by neighbor-joining method revealed the position of T6-T9 in the species of Trichoderma (Fig. 1). Based on the molecular and morphological characteristics (Yang 2009), the four Trichoderma isolates T6-T9 (GenBank accession no: OQ727252; OQ727298; OQ727299; OQ727300) were identified as T. asperellum.

In vitro antagonistic ability of VOCs of Trichoderma isolates against B. cinerea

In the mycelial growth inhibition assay (Fig. 2a), VOCs produced by the nine Trichoderma isolates significantly reduced mycelial growth of the pathogen compared with the control (Fig. 2b), the inhibition percentage of the nine Trichoderma isolates ranged from 13.9–63.0%. The highest inhibition percentage belonged to T7 (63.0%), showing a significant inhibition than T1 (53.0%), T2 (51.8%), T3 (51.0%), T4 (38.1%), T8 (35.6%), T9 (32.4%), T6 (25.0%) and T5 (13.9%).

In the conidial germination inhibition assay(Fig. 3), twenty four hours after inoculation, the conidial germination of B. cinerea was significantly inhibited by the VOCs emitted by the nine Trichoderma isolates compared with the control. Similarly, the highest inhibition percentage belonged to T7 (96.3%), showing a significant inhibition compared with T3 (68.0%), T9 (66.4%), T8 (57.7%), T4 (45.3%), T2 (37.0%), T6 (36.9%), T5 (32.6%) and T1 (17.6%).

Effect of VOCs of T7 on the plasma membrane of B. cinerea

The integrity of the plasma membrane of B. cinerea were observed under fluorescence microscope (Fig. 4). Compared with control (Fig. 4a), conidia germination of the pathogen was inhibited by VOCs produced by Trichoderma T7, therefore, there was no mycelia observed (Fig. 4b). Conidia, especially the young conidia treated with VOCs showed red fluorescence, flattened and slightly concave, while the bigger and older conidia showed plump and intact plasma membrane, thus they did not show any red fluorescence. Similarly, there was no red fluorescence in conidia and mycelia in controls (Fig. 4a). Therefore, it is inferred that the VOCs from T7 damaged the permeability and integrity of plasma membrane in young cells of B. cinerea, resulting in cytoplasm leakage.

Antagonistic activity of VOCs from T7 against gray mold on strawberry fruits

Antagonistic activity of VOCs on gray mold on strawberry fruits was assessed according to disease index and biocontrol efficacy. Three days after inoculation, strawberry fruits inoculated only with B. cinerea was diseased, while VOCs-treated fruits (B.c + T7) remained healthy. Six days after inoculation, soft rot symptoms appeared on the VOCs-treated fruits. Seven days after inoculation, all the fruits inoculated only with the pathogen exhibited severe soft rot and a large amount of aerial mycelial masses appeared on the decayed tissues (Fig. 5a, B.c-only), the disease index was up to 71.9 (Fig. 5b, B.c-only). In contrast, in presence of Trichoderma T7, fruits rot were significantly inhibited by the VOCs, no aerial mycelia appeared on the rot tissues (Fig. 5a, B.c + T7), the disease index was 16.7, resulting in 76.9% biocontrol efficacy (Fig. 5b, B.c + T7). While the injured fruits treated only with T7 remained healthy (Fig. 5a, T7-only).

Antagonistic activity of VOCs from T7 against gray mold on strawberry leaves

In this assay, two days after inoculation, leaf spot symptoms were observed in the leaves inoculated with B. cinerea, eight days after inoculation, the disease index of the treatment was up to 70.8 (Fig. 6b, B.c-only), a large amount of mycelia, conidia and conidiophores appeared on the spots (Fig. 6a, B.c-only), while no symptoms appeared on the leaves treated with VOCs of T7 and the pathogen (Fig. 6a, B.c + T7), the biocontrol efficacy was up to 100% (Fig. 6b, B.c + T7), so far as to 15 days after inoculation, the trial leaves turned yellow and dry-up. Similarly, the injured leaves treated only with T7 remained healthy (Fig. 6a, T7-only).

Antagonistic activity of VOCs from Trichoderma T7 against B. cinerea on strawberry leaves

In the staining assay, leaves inoculated with B. cinerea for 8 days was used. In presence of VOCs from T7, just like on WA, most conidia of the pathogen on strawberry leaves was inhibited and wasn’t able to germinate, conidia surface was flattened and concave, resulting in obvious plasmolysis and cytoplasm leakage (Fig. 7a), though a small amount of conidia was able to germinate, the hyphae was very short and the infection pads was invisible (Fig. 7b) as compared to control, of which all the conidia germinated and a large number of infection pads were observed (Fig. 7c).

In recent years, the production of VOCs by fungi has become one of the hotspots in biocontrol research due to their small size and easy diffusion (Inamdar et al. 2020). They regulate the interactions between different ecological niches, participating in host recognition, defense and competition (Hung et al. 2015). Trichoderma, a well-known fungal biocontrol agent, has massive potential for biocontrol as its VOCs can combat pathogens without direct contact and quickly diffuse and spread in enclosed environments to reach infected fruit habitats (Harman et al. 2004; Karsli et al. 2021; Bello et al. 2022). In this study, all the nine Trichoderma isolates shared significantly high activity against B. cinerea in vitro compared to control, among them, T. asperellum T7 was a good biofumgant against B. cinerea because of the best inhibition percentage to mycelial growth (63.0%) and conidial germination (96.3%) of the pathogen, which acted by damaging the integrity of the plasma membrane of pathogenic conidia by VOCs produced by T7, and resulting in cytoplasm leakage.

Numerous studies have shown that VOCs produced by many BCAs have significantly antagonistic activity against various post-harvest disease on fruits and vegetables (Jiang et al. 2018; Don et al. 2021; Zhong et al. 2021; Zou et al. 2021; Bello et al. 2022; You et al. 2022). Among them, more attention has been paid to B. cinerea due to the disease severity caused by it. For examples, VOCs produced by Pseudomonas fluorescens ZX, which inhibited spore germination and mycelial growth of B. cinerea, reduced the disease incidence in grape fruits from 99–73% (Zhong et al. 2021). VOCs from Bacillus velezensis 5YN8 and DSN012 had a preventive effect over 50% against pepper gray mold (Jiang et al. 2018). Trichoderma atroviride IC-11 VOCs reduced the spore germination rate of B. cinerea by 70% and reduced the incidence of gray mold on blueberry fruits by 83% (Bello et al. 2022). The VOCs produced by Trichoderma koningiopsis T-51 had high inhibitory activity against the mycelial growth and spore germination of B. cinerea and Fusarium oxysporum, and had a preventive effect of up to 50% against tomato fruit rot (You et al. 2022). The volatile substances of Streptomyces platensis F-1 show prominently in vivo antifungal effects against Rhizoctonia solani of rice, Sclerotinia sclerotiorum of oilseed rape, and B. cinerea of strawberry, inhibiting their growth by 60 − 70% (Wan et al. 2008). As for Botrytis gray mold occurred on strawberry fruit, Huang et al. (2011) found VOCs produced by Candida intermedia C410 significantly reduced the incidence and severity of gray mold on strawberry, VOCs of C. intermedia C410 were promising biofumigants for control the disease on strawberry. In vivo assay showed that Hanseniaspora uvarum reduced B. cinerea infection of strawberry and maintained fruit appearance, firmness and total soluble solids via VOCs production, significantly controlled postharvest gray mold of strawberry and prolonged the storage time and shelf life (Qin et al. 2017). Similarly, VOCs produced by yeast isolates Scheffersomyces spartinae W9 reduced the disease spot diameter of strawberry fruit surface by 45.9% (Zou et al. 2021). In our work, VOCs produced by T. asperellum T7 significantly reduced the disease incidence, and gave 76.9% and 100% biocontrol efficacy against gray mold on strawberry fruits and detached leaves, respectively, which acted by damaging the plasma membrane of B. cinerea and resulting in cytoplasm leakage. These results suggested that VOCs produced by T7 was a promising biofumigants not only for controlling post-harvest Botrytis fruit rot but also for controlling pre-harvest strawberry gray mold in a relatively enclosed environment.

Postharvest fruits of strawberry are highly perishable, which provides conditions for fungal pathogen infection, in fact, most postharvest fruits of strawberry cannot be stored overnight (Lan 2022). Thereby, numerous fungicidal methods combined with low-temperature storage were used to reduce postharvest fruits of strawberry waste caused by pathogenic fungi, prolonging the fruit shelf life (Qin et al. 2017; Lan 2022). In this work, strawberry fruits inoculated only with B. cinerea were diseased 3 days after inoculation, while the VOCs-treated fruits were diseased 6 days after inoculation. That is to say, VOCs produced by T7 can prolong the fruit shelf life by 3 days in presence of B. cinerea. This result had significant implications for strawberry preservation and industry.

Volatile metabolites produced by Trichoderma spp. have been shown to contain phenylethanol (PEA), which has potent inhibitory effects against fungal species such as Candida spp. and Aspergillus spp. (You et al. 2022). Also, lactone 6-Pentyl-2H-pyran-2-one (6PP) is another common metabolite produced by Trichoderma spp., which has antagonistic activity against several pathogens, including R. solani, F. oxysporum and B. cinerea (Vos et al. 2015). VOCs produced by T. asperellum T7 had strong antifungal activity, which inhibited the pathogen’s mycelia growth and damaged the conidial plasma membrane, resulting in cytoplasm leakage. Obviously, specific bioactive metabolites in the VOCs played a crucial role in inhibiting strawberry gray mold. However, the chemical compositions of the VOCs remained unknown in our work.

The results of this work revealed that the VOCs produced by the nine isolates of Trichoderma significantly inhibited mycelial growth and conidial germination of B. cinerea in vitro, among them, VOCs of T. asperellum T7 had the best antagonistic activity against the pathogen, shared 76.9% and 100% biocontrol efficacy against gray mold on strawberry fruits and on detached leaves, respectively, prolonged the fruit shelf life by 3 days in presence of B. cinerea, completely protected the leaves from the pathogen infection, which acted by damaging the pathogen’s plasma membrane and resulting in cytoplasm leakage.

Based on the above mentioned assays, the chemical compositions of VOCs produced by T. asperellum T7 implicated are further required to fully evaluate in order to understand their roles, the impact on human health and non-target organisms.

Compliance with ethical standards This manuscript has not been submitted to any other journal for simultaneous consideration. All the co-authors consented to the manuscript submission and have contributed sufficiently to the scientific work and therefore share collective responsibility and accountability for it. This work does not contain any studies with human participants or animals performed by any of the authors.

Conflicts of interest The authors have no conflict of interest to declare that are relevant to the content of this article.

Acknowledgements All the co-authors would like to thank the TaiShan Scholar Construction Foundation of Shandong Province and the Research Foundation for Advanced Talents in Qingdao Agricultural University (630901) for financial fundings.

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Editorial decision: Major revisions
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03 Jul, 2023
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You are reading this latest preprint version

Biocontrol of strawberry Botrytis gray mold and prolong the fruit shelf-life by fumigant Trichoderma spp.

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Abstract

Figures

Introduction

Materials and methods

Fungal strains and growth conditions

Molecular identification of Trichoderma isolates

In vitro antagonistic activity of VOCs of Trichoderma isolates against B. cinerea

Inhibition of mycelial growth of B. cinerea

Inhibition of conidial germination of B. cinerea

Damage to the plasma membrane of B. cinerea caused by VOCs of Trichoderma T7

Antagonistic activity of VOCs produced by T7 on gray mold on strawberry fruits

Antagonistic activity of VOCs produced by T7 on gray mold on strawberry leaves

Antagonistic activity of VOCs from Trichoderma T7 against B. cinerea on strawberry leaves

Statistical Analysis

Results

Molecular characteristics of Trichoderma isolates

In vitro antagonistic ability of VOCs of Trichoderma isolates against B. cinerea

Effect of VOCs of T7 on the plasma membrane of B. cinerea

Antagonistic activity of VOCs from T7 against gray mold on strawberry fruits

Antagonistic activity of VOCs from T7 against gray mold on strawberry leaves

Antagonistic activity of VOCs from Trichoderma T7 against B. cinerea on strawberry leaves

Discussion

Conclusions

Declarations

References

Status:

Version 1