1. Introduction
Alternaria alternata is a devastating necrotrophic fungus with a high incidence and causes brown spots on many plant leaves and fruit, including citrus, cherry tomato and tobacco [
1,
2,
3]. This notorious fungal pathogen is a common aggressive fungus that can sense specific stimuli, colonize leaves or fruit and over-secrete unique host-selective toxins in plants, eventually causing diseases on different host plants [
4]. At the initial stage,
A. alternata infection results in small yellowish-brown round spots on plants and then the spots gradually increase in size into bigger ones with the occurrence of decay, which results in a considerable reduction in plant yield and quality [
5,
6]. Specifically, tobacco brown spot disease, a destructive foliar disease caused by
A. alternata, is a crucial fungal disease endangering tobacco yield that infects most of China’s tobacco-growing fields. Currently, the most effective treatment for this disease is the application of traditional fungicides such as dimetachlone and mancozeb [
3]. Unfortunately, these conventional fungicides gradually lead to increasingly serious environmental pollution and food security issues, which can be reversed by effective, biodegradable and safe natural fungicides [
7].
Investigations have indicated that biopesticides can protect plants from pathogens and increase crop yields without adverse environmental impacts, so they are an appropriate alternative approach to traditional pesticides and can reduce the use of agricultural fungicides [
8]. Natural antagonistic microorganisms for bio-control, including
Bacillus,
Pseudomonas and
Streptomyces, are abundant in soil and can support plants not only by controlling plant pathogens but also by increasing the soil nutrient content [
9,
10]. In this context,
Streptomyces, acting as Gram-positive bacteria, are significant bio-control agents and mainly thrive in decaying organic matter and soil. As bacteria of the genus Actinomyces,
Streptomyces have slender hyphae without multi-core or isolated branches. Up to now, many
Streptomyces have been used to produce various secondary metabolites, which are generally regarded as agro-antibiotics and antibiotics [
11]. With the high relevance of
Streptomyces in controlling many fungal pathogens, the use of its strains as bio-control agents for plant diseases is promising and has been popularized and commercialized in numerous areas and countries [
12]. Many Actinomycete species, particularly the genus
Streptomyces, can be used to control some pathogenic fungi. However, little is known about the antifungal effect of
Streptomyces hygroscopicus (
S.
hygroscopicus) against the plant pathogen
A. alternata.
In this study, an isolation of S. hygroscopicus was obtained from rhizospheric soil and named JY-22. The strain of S. hygroscopicus JY-22 was further used to verify its antagonistic antifungal properties and mechanism against A. alternata. Malondialdehyde (MDA) content, ergosterol content, soluble protein content and the integrity of the cell membrane of A. alternate, which are very important for the survival of eukaryotic microbes, were used to investigate the inhibition mechanisms of S. hygroscopicus against A. alternate. Its ability to control tobacco brown spot caused by A. alternata was also further evaluated in detached-leaf and field tests.
2. Materials and Methods
2.1. Antagonistic Actinomycetes and Pathogen Sources
The Streptomyces hygroscopicus strain, which was named JY-22 and used in the experiment, was isolated from the soil of Jinyun Mountain (29°41′ N, 106°18′ E, 895 m elevation, Chongqing, China). A. alternata (Alternaria alternata (Freis) Keissler) was isolated from naturally infected tobacco, provided by Professor Dou Yanxia, College of Plant Protection, Southwest University, and was identified as a highly pathogenic strain.
2.2. Plant Culture
Tobacco seeds (the cultivar “K326”) were germinated in a growth chamber at a relative humidity of 60% and a temperature of 25 °C. Seedlings at the same stage were transferred into new pots filled with potting medium (Pindstrup Mosebrug A/S, Denmark) and incubated in a growth chamber at 22 °C with a cycle of 16 h light and 8 h dark [
13]. The seedlings were used in experiments until 5–6 truly fully expanded leaves were observed. A total of 20 tobacco plants were used for each treatment. The pots were drenched with the same volume of deionized water to maintain the moisture content at 60%
w/
w. Each experiment was repeated three times.
2.3. In Vitro Antagonistic Assay
The ability of antagonistic microorganisms (S. hygroscopicus JY-22) to inhibit the mycelial growth of A. alternata in vitro was detected by the confrontation culture method with minor modifications. Here, a slide (7.5 cm × 2.5 cm) was placed in every petri dish, and PDA medium was added to the plates until it sufficiently flooded the slides. S. hygroscopicus JY-22 (JY-22) was inoculated onto PDA medium on one side of the slide. After three days of incubation, A. alternata was inoculated on the other side of the slide. The plates containing A. alternata only served as a control. Each experiment was repeated three times, and plates were incubated in an incubator at 28 ± 2 °C for 7 days. Finally, the morphological changes of antagonistic microorganisms and A. alternata JY-22 were analyzed using a light microscope.
2.4. Preparation of Culture Filtrate
JY-22 was inoculated into sterilized PDA medium and cultivated in an incubator at 28 ± 2 °C for 4 days. Circular mycelial blocks (Φ = 8 mm) were obtained by punching at the edges of colonies with a hole punch. Then, the S. hygroscopicus blocks were inoculated into Erlenmeyer flasks containing sterilized broth medium (1 L sterilized medium contained glucose (20 g), cornmeal (15 g), soybean flour (30 g), NaCl (2.5 g) and CaCO3 (2 g)). On the other hand, the pure sterilized broth medium (uninoculated) served as the control group.
All cultures were placed in a shaker incubator at 28 °C and 160 r/min for 72 h. The supernatant was collected by centrifugation at 12,000 r/min, and the medium residue was removed. This fermented liquid was filtered by a 0.22 μm drainage pin-type filter to remove impurities and obtain the culture filtrate. The JY-22 culture filtrate was kept at 4 °C before use.
2.5. JY-22 Culture Filtrate Antagonistic Assays
To investigate the antibacterial activity of the JY-22 culture filtrate, the culture filtrate was diluted with pure sterilized broth medium to different proportions ranging from 10 to 100 times (10, 20, 40, 80 and 100 times), and the relative inhibitory effect of the fermentation broth on the growth of an
A. alternata colony was measured by a typical method [
14]. Specifically, the culture filtrate suspension was added to the solid PDA medium (50 °C) at the final test concentrations mentioned above. The pure sterilized broth medium added to solid PDA medium was the control group. Next, the
A. alternata block (Φ = 0.45 mm) was inoculated in the center of a PDA plate and incubated at 28 °C for 7 days. The colony diameters on agar plates were measured. The fungal relative inhibition rate was calculated by the following formula:
where A represents the control colony diameter and B represents the experimental colony diameter.
2.6. Inhibition of JY-22 Culture Filtrate on Spore Germination
To further explore the antagonistic ability of JY-22 culture filtrate on the spore germination of
A. alternata, the inhibitory rate of germination was investigated. Briefly, 100 μL
A. alternata spore suspension (2.0 × 10
5 CFU/mL) was added to a 96-well plate. Then, 100 μL fermented filtrate was diluted with pure sterilized broth medium and added to the 96-well plate for final dilution (10, 20, 40, 80 and 100 times). Meanwhile, 100 μL pure sterilized broth medium was added into the 96-well plate to obtain the control group. After being mixed completely, the test substances contained
A. alternata spores and JY-22 culture filtrates; however, in the control group, only the
A. alternata spores were present. Every experiment was repeated three times. After incubation at 28 °C for 36 h, the mixture was examined microscopically. The relative inhibition rate was calculated by the following formula:
where A represents the number of germinated spores in the control group, and B represents the number of germinated spores in the experimental group.
2.7. Measurement of Extracellular Conductivity
Referring to a previous study [
15], the membrane permeability was measured using the extracellular conductivity method with some modifications. Specifically, an
A. alternata block (Φ = 0.45 mm) was inoculated in medium and shaken at the speed of 160 r/min at 28 °C for 36 h. JY-22 culture filtrate was added to conical flasks containing
A. alternata mycelia at diluted concentrations of 10, 20, 40, 80 and 100 times, respectively. The
A. alternata mycelia treated with pure sterilized broth medium in conical flasks served as the control group. After the mycelia were treated with JY-22 culture filtrate in shakers for differing lengths of time (0, 30, 60, 90, and 120 min), the extracellular conductivity was measured by a conductivity meter (Shanghai Precision Scientific Instrument Co., Ltd., Shanghai, China). Every experiment was repeated three times.
2.8. Assays of Malondialdehyde (MDA)
The MDA assay was carried out according to a previous method [
16] with minor modifications. Briefly, after treatment with JY-22 culture filtrate or pure sterilized broth medium (control group) as described in
Section 2.6 above, the mycelia were cultivated for 96 h and separated by centrifugation. Next, the mycelia were gently washed with distilled water and the excess water was soaked up by filter paper. Then, the dried mycelia (2.0 g) were mixed with 2 mL trichloroacetic acid (20%,
w/
v) and barbiturate (0.5%,
w/
v) and ground adequately. The mixture was subsequently transferred into a centrifuge tube for centrifugation for 10 min (10,000 r/min). Next, the supernatant was boiled for 10 min. After cooling, the MDA content of
A. alternata was calculated by measuring the absorbance of the supernatant at 450 nm, 532 nm and 600 nm. Every experiment was repeated three times. The formula was as follows:
2.9. Determination of Ergosterol Content
The ergosterol content of
A. alternata was investigated as previously described [
17]. First,
S. hygroscopicus culture filtrate was added to flasks containing
A. alternata mycelia suspensions at the final test concentrations (10, 20, 40, 80 and 100 times for the JY-22 culture filtrate). In the control group, pure sterilized broth medium was used instead of JY-22 culture filtrate. After the treated
A. alternata was cultured for 96 h, the mycelia were washed twice with distilled water and harvested. Then, the mycelium samples were gently dried with filter paper to avoid affecting the measurement of ergosterol in the plasma membrane. Next, the mycelium samples were added to methanol/trichloromethane at a ratio of 2:1, ground with liquid nitrogen and cooled at room temperature for 1 h. The samples were transferred to a mixture of sterile distilled water (6 mL), trichloromethane (6 mL) and phosphate buffer (6 mL) and incubated at 80 °C for another 4 h. Methanol (8 mL) and ethanol (2 mL) were added to these samples and mixed for saponification at 60 °C for 1 h. Then, petroleum ether (15 mL) was added, and the resulting solution was shaken for 20 min and extracted twice. Subsequently, the upper liquid was washed with sterilized water and the supernatant was removed and diluted with ethanol (95%) to 10 mL. Finally, the absorbance was measured at 282 nm by a UV/VIS spectrophotometer (TU-19, Beijing Purkinje General Instrument Co, Ltd., Beijing, China). Samples without the culture filtrate treatment were used as the control group. Every experiment was repeated three times. The formula to determine the inhibition rate of ergosterol was as follows:
where A represents the optical density of the control group, and B represents the optical density of the experimental group.
2.10. Soluble Protein Content Measurement
The proteins released into the supernatant were defined as soluble proteins, which usually reflect the integrity of cells. Here, the concentrations of soluble protein were assessed using the Bradford method (1976), using bovine serum albumin as standard. [
18]. Briefly, the mycelia were treated with different concentrations of JY-22 culture filtrate or pure sterilized broth medium (control group), and the protein contents were determined by measuring the optical density (OD
595). The crude protein extracts were centrifuged at 12,000 rpm for 15 min at 4 °C. Next, the binding of the proteins and Coomassie Brilliant Blue was measured at 595 nm by a spectrophotometer (Beijing Purkinje General Instrument Co, Ltd., China). The experiment was repeated three times.
2.11. Detached-Leaf Assays
This experiment was performed to determine the protective effect and curative effect of the culture filtrate. An amount of 2 mL JY-22 culture filtrate (the final test diluents were 10, 20, 40, 80 and 100 times) or 50% carbendazim was sprayed onto tobacco leaves. The same volume of pure sterilized broth medium was used for control treatment. At 24 h before or after spraying, each leaf was inoculated with an A. alternata block. After cultivating in a plant growth chamber at a temperature of 25 °C and a relative humidity of 85–90%, the stalks were drenched with moist gauze to stay hydrated. After 5 days, the control effect of JY-22 culture filtrate on A. alternata in vivo was observed. Every assay was performed three times.
2.12. Field Experiments
The field experiment was carried out in Fuling, Chongqing, China (29°43′3.61″ N and 107°36′11.45″ E). The tobacco cultivar K326, which is susceptible to A. alternate, was planted to test the control effect of JY-22 culture filtrate against A. alternata. The fertilizer was applied following a standard, and all plants were cultivated under the same amount of well water irrigation in the field management. During the entire growth period, no fungicides were used except for the experimental treatment. The field experiments were conducted to determine protective and curative effects as follows: Protective effect: Tobacco plants were sprayed with JY-22 culture filtrate, 50% carbendazim or well water (control group). After 24 h of spraying, an A. alternata spore suspension (2.0 × 105 cfu/mL) was inoculated onto tobacco plants by the spraying method. Disease indexes for three different treatments were calculated to evaluate the protective effect. Curative effect: After inoculated with A. alternata for 24 h, tobacco was sprayed with the JY-22 culture filtrate or well water (control group) to evaluate the curative effect. The disease severity was evaluated for each treatment. The experiments were performed as randomized blocks with four replicates. The field experiment was repeated in 2020, 2021 and 2022.
2.13. Data Analysis
All experiments included at least three independent replicates. Statistical differences were estimated by SPSS 19.0 using independent-sample t-tests for comparison between two treatments or one-way analysis of variance (ANOVA) for multiple treatments. All data were recorded as the mean ± standard deviation with * and ** representing statistical significance at the levels of p < 0.05 and p < 0.01, respectively.
4. Discussion
Recently, plant fungal disease has caused massive threats and losses to agricultural productivity worldwide. Currently, due to the abuse of fungicides and continuous cropping, plant diseases have become a great threat to human survival. Moreover, other great threats, such as antibiotic resistance of pathogens and environmental risk factors caused by the inappropriate application of fungicides, have emerged. Thus, eco-friendly and effective agents that control fungi are urgently needed, and biological control has attracted extensive research interest in the sustainable management of fungal plant diseases [
12,
14,
19]. Usually, the main method underlying bio-control to suppress plant disease is to utilize antagonistic living organisms to defeat plant pathogens [
20].
As bio-control agents, Actinomycetes have been found to have promising effects on a number of crop diseases [
9]. Therein,
Streptomyces hygroscopicus, as a vital Actinomycete, inhibits the growth of target microorganisms because of its robust viability and can utilize various compounds as the sole C-source, efficiently competing for space and nutrients [
9,
21]. In agricultural and horticultural protection,
Streptomyces spp. produce more than 60% of antibiotics that serve as inhibitors of phytopathogens [
21]. Hence, it is still crucial to develop bio-control agents and study their antifungal activities.
The bio-control strain in our study (Actinomyces strain JY-22) was identified as
S. hygroscopicus. Pathogen infection and reproduction proceed with mycelium growth, owing to the asexual reproduction of mycelium [
22]. Inhibiting fungal growth is a typical method to reduce fungal proliferation. Dual culture experiments showed that JY-22 and
A. alternata competed for limited space and nutrients, thereby interfering with pathogen growth. In contrast, at the ultrastructural level, we observed obvious morphological alterations of
A. alternata hyphae by light microscopy and revealed that the JY-22 strain had strong antifungal activity against
A. alternata by curbing or damaging the hyphae. The strong activities of the chitinase, cellulase and β-1,3-glucanase of
S. hygroscopicus could destroy or disintegrate the cell walls of fungal pathogens [
23,
24]. Furthermore, the injuries might have caused the cellular breakdown of
A. alternata, which undoubtedly affected the survival of the fungus.
Actinomycetes as bio-control agents are usually directly applied to plants through spore suspensions or culture filtrates [
25]. More importantly, researchers have speculated that the key factors contributing to the antifungal activities of this bio-control microbial strain are metabolites [
9,
12]. Hence, the antifungal activities of
S. hygroscopicus fermentation filtrate on both mycelial growth and spore germination have been primarily assessed. All tested JY-22 culture filtrate diluents (at concentrations of 10–100 times) exhibited marked antifungal effects against
A. alternata. Therefore, these observations confirmed that both JY-22 and its fermentation filtrate can inhibit the spore germination and mycelial membrane formation of
A. alternata. Some possible mechanisms underlying the antifungal properties of JY-22 culture filtrate against bio-control strains have been proposed. Previous reports have disclosed that
S. hygroscopicus is an efficient bio-control agent because it produces a large number of secondary metabolites. In this case, antibiotic production is one of the key functions, including various antimicrobial compounds [
14].
S. hygroscopicus could produce approximately 650 types of secondary bioactive metabolites, some of which were applicable as antimicrobial agents to combat pathogenic bacteria in plants [
25]. Among these bioactive metabolites, rapamycin and clethramycin have antifungal activity [
26,
27]. Therefore, according to these works within the literature, metabolites from the JY-22 culture filtrate are speculated to some possess some antibiotic properties. Moreover, microbes produce abundant secondary metabolites during the fermentation process, and these metabolites may change with a change in environment and further suppress pathogen growth [
27]. Herein, it was speculated that one or more metabolites produced in the fermentation process of JY-22 can inhibit
A. alternata growth. However, the exact mechanism needs to be investigated.
Furthermore, several studies have shown that the mechanism of antifungal agents against plant pathogens is related to membrane disruption by some highly lipophilic and low-molecular-weight components that can pass through the cell membranes easily and lead to intracellular leakage, eventually resulting in fungal cell death [
12,
28,
29]. As expected, the extracellular conductivity and ergosterol and MDA contents in our study conform to the above mechanism. The conductivity of the group incubated with JY-22 culture filtrate was higher than that of the control group during the experiment, and the values increased with time and fermentation concentration. Moreover, ergosterol is a major sterol component of the cell membrane, vital to cell integrity and normal function [
17]. JY-22 culture filtrate exhibited a strong inhibition on ergosterol content for 10–80 times diluents, revealing that it had strong permeation and cell membrane disruption. The results of this study were similar to those of recent studies on plant-derived antifungal agents [
15]. Collectively, the physical and morphological structures of
A. alternata hyphae were changed after treatment with JY-22 culture filtrate, largely attributed to the effect of the filtrate on the permeability and integrity of the pathogen’s cell membrane. In addition, JY-22 culture filtrate resulted in mass protein loss from cells, further indicating irreversible damage to cytoplasmic membranes.
In the detached-leaf and field tests, the disease severity index (DSI) in the control group reflected that the tobacco plants were under disease pressure. Tobacco brown spot is a leaf disease, and a reduction in this disease means an increase in tobacco production. Hence, these results further suggested that JY-22 culture filtrate was effective against tobacco brown spot disease, suggesting that it is a promising bio-control agent against
A. alternata. Interestingly, our data demonstrated that the preventative treatment of the culture filtrate had a better control effect than the curative treatment. Previous studies suggested that with the application of the fermented organic fertilizer
S. hygroscopicus B04, the B04 strain could better adapt to the strawberry rhizosphere and protect plants from pathogens [
30]. In this regard, JY-22 culture filtrate might have potential applications as an accelerator to increase the colonization of some other
S. hygroscopicus bio-control strains, eventually increasing fungal densities and inhibiting pathogens. In addition to its broad-spectrum antifungal ability, which includes activity against
Fusarium oxysporum,
S. hygroscopicus can also be extensively used in crop production and has economic development benefits due to its advantages in plant and fruit growth and production promotion [
9,
30,
31]. Overall, the culture filtrate of
S. hygroscopicus may be a potential bio-control agent against tobacco brown spot disease. Nevertheless, some challenges remain, including determination of its effective components, specific antifungal mechanisms and safety during usage in plant protection.