Biocontrol Activity of Alternaria angustiovoidea Against Trichothecium roseum Through Inhibiting Its Growth, Pathogenicity, and Gene Expression

Abstract

Apple mold heart disease, primarily caused by Trichothecium roseum, is the most severe disease affecting stored apples. Developing biocontrol resources as an alternative to chemical pesticides is crucial for the advancement of green agriculture. This study demonstrated that a pathogenic fungus isolated from sunflower leaves exhibited specific inhibitory effects against T. roseum. Through morphological observation, identification, and the construction of a phylogenetic tree analysis, the fungus was identified as Alternaria angustiovoidea. GW2A was found to inhibit the spread of diseases on apple twigs. Additionally, GW2A has significant preventive and therapeutic effects on apple mold heart disease. Furthermore, GW2A can induce apple trees to upregulate defense-related genes, thereby enhancing resistance. Transcriptome analysis revealed that GW2A inhibits T. roseum growth by suppressing the function of polysaccharide hydrolases, oxidoreductases, and intermediate steps in carbohydrate metabolism. In conclusion, our research has effectively isolated and characterized A. angustiovoidea, revealing its significant potential as a biocontrol agent against T. roseum and apple mold heart disease, particularly in areas where sunflowers and apple trees are not co-cultivated. Additionally, we demonstrated its ability to induce resistance in apple trees, offering a sustainable approach to disease management in apple cultivation.

1. Introduction

Plants in nature are continually subjected to invasion by various pathogenic organisms. Chemical control, though effective in agriculture, has significant environmental drawbacks [1]. One of its disadvantages is environmental contamination, as pesticides can leach into soil and waterways, harming aquatic life and disrupting ecosystems [2,3]. Moreover, these chemicals can accumulate in the food chain, posing risks to human and animal health via bioaccumulation [4,5]. Additionally, non-target organisms, such as beneficial insects and wildlife, may be adversely affected, thereby disrupting natural biodiversity [3,6,7,8]. Biological control substantially mitigates the negative impacts of pesticides. It inhibits pathogen invasion and enhances plant resistance [9]. Although the effectiveness of biological control is highly influenced by abiotic factors such as temperature, humidity, and soil conditions, ensuring its environmental stability remains a significant challenge [10]. As modern agriculture advances, biological control becomes increasingly crucial for plant protection [11].

Numerous microorganisms have been engineered as biocontrol agents [12,13]. Among these, Trichoderma is a widely distributed, broad-spectrum biocontrol fungus. Specifically, the secondary metabolites of Trichoderma inhibit the spread of Phytophthora-caused plant diseases [14,15]. Additionally, Trichoderma virens and Trichoderma atroviride can inhibit the growth of Rhizoctonia solani through predation and parasitism, respectively [16]. In addition to Trichoderma, Pseudozyma flocculosa produces flocculosin to induce cell collapse in Pythium ultimum, thereby controlling powdery mildew [17]. Similarly, Pseudomonas chlororaphis secretes antibiotics, siderophores, and multiple organic compounds to suppress the invasion of various pathogens [18]. Furthermore, the endophytic antagonistic fungus Chaetomium spirale, isolated from Populus tomentosa, can inhibit the mycelial growth of Valsa ceratosperma [19].

Certain pathogenic microorganisms can function as biocontrol agents. For example, Sclerotinia sclerotiorum, a notorious pathogenic fungus with a broad host range, can grow in wheat and resist the invasion of stripe rust and Fusarium head blight [20,21,22]. Alternaria spp., widely found in nature, are a primary cause of plant diseases in crops, cash crops, and fruit trees. However, some species possess biocontrol potential and can serve as biocontrol agents. Alternaria alternata (Fr.) Keissler can parasitize Puccinia striiformis f. sp. tritici (Pst), thereby limiting wheat stripe rust disease spread [23]. A. basellae can induce mycelial deformity and protoplast exosmosis in Valsa ceratosperma, demonstrating significant inhibitory effects on apple canker in the field [24].

China, the world’s leading producer of apples, has maintained a stable apple planting area at a high level over recent years. According to publicly released information (National Bureau of Statistics, China, 2024) [25], China’s apple production has consistently exceeded 49.6 million tons, leading globally and holding a significant share in the international apple market. The ongoing development of the apple industry has made apples a vital part of China’s agricultural economy, contributing substantially to the increase in agricultural output value.

Apple mold heart disease, affecting harvested apples, is caused by a combination of multiple fungal infections, with T. roseum being the predominant pathogen [26]. This disease leads to preharvest fruit drop, core rot and mold in mature fruits, and decay during storage, severely impacting the economic value of harvested apples [27,28,29]. Additionally, T. roseum produces mycotoxins like trichothecenes, posing risks to human and animal health [30,31]. The adoption of biocontrol measures for apple mold heart disease reflects a growing trend in green agriculture. Currently, few biocontrol agents are available for postharvest apple mold heart disease. Lactobacillus plantarum C10 shows strong antifungal activity towards T. roseum, and it can induce defense responses in muskmelon (Cucumis melo L.) fruit [32]. Sodium propylparaben (spp.) can disrupt the cell membrane and wall of T. roseum, thereby reducing the incidence of muskmelon pink rot [33]. Two novel cationic antifungal peptides, P-1 and P-2, extracted from Bacillus pumilus HN-10, demonstrate efficacy against T. roseum [34].

Biological control contributes to sustainable agriculture by harnessing natural enemies, such as predators, parasitoids, and microbes, to suppress pest populations, thereby reducing dependency on chemical pesticides and mitigating their adverse environmental and health impacts. This eco-friendly approach supports biodiversity, enhances ecosystem resilience, and aligns with global efforts to achieve sustainable food production [35,36].

Therefore, there is an urgent need to develop and explore novel biocontrol agents to mitigate the loss of apple quality and yield during storage, addressing the growing challenge of postharvest mold heart disease in apples. This study identified an Alternaria strain derived from sunflower leaves exhibiting significant antifungal activity against Trichothecium roseum and preliminarily elucidated its biocontrol mechanisms, thereby providing a promising and innovative approach for the management of postharvest fruit diseases.

2. Materials and Methods

2.1. Fungi and Plant Materials

Strains of T. roseum Tr1, Diaporthe spiculosa ZJ13, Magnaporthe oryzae Guy11, Rhizoctonia solani AG-1 IA YN-1, A. tenuissima DT1828-A, and Colletotrichum gloeosporioides HGUP0011 were identified and are currently stored in the College of Horticulture, Agricultural and Forestry Engineering Research Institute, Ludong University.

The fungal strain T. roseus Tr1 was isolated from apple fruits (Malus pumila Mill.) collected in the Fushan District, Yantai County, Shandong Province, China. Similarly, Diaporthe spiculosa ZJ13 and Alternaria tenuissima DT1828-A were obtained from cherry fruits (Prunus avium “Mei Zao”) in the same region. Additionally, Colletotrichum gloeosporioides HGUP0011 was isolated from pear fruits (Pyrus sp.) in Laiyang, Yantai County, Shandong Province, China. The strains Magnaporthe oryzae Guy11 and Rhizoctonia solani AG-1 IA YN-1 were maintained in the laboratory of the College of Horticulture, Agricultural and Forestry Engineering Research Institute, Ludong University.

Diseased plant tissues were excised into small sections (5 mm × 5 mm) at the junction of diseased and healthy tissues. The surfaces of these tissues were disinfected with 75% ethanol for 30 s, followed by three rinses with sterile water. Subsequently, the tissues were treated with 10% sodium hypochlorite (NaClO) for 3 min and then thoroughly rinsed with sterile ultrapure water. The sterilized tissues were then plated onto potato dextrose agar (PDA; Hangzhou Microbial Reagent Co., Ltd., Yuhang District, Hangzhou, China) and incubated at 25 °C in the dark for one week.

The fungi genomic DNA was isolated utilizing the DNALyse Amplification Kit (CW0556S, CWBIO, Changping District, Beijing, China). Following a 5-day cultivation on potato dextrose agar (PDA) at 28 °C, fungal mycelia were aseptically harvested using a sterile surgical blade and transferred into a 1.5 mL sterile microcentrifuge tube. Subsequently, 200 μL of preheated lysis buffer (DNALyse Amplification Kit, CWBIO, Changping District, Beijing, China) was added to the biomass. The mixture underwent sequential thermal treatments: primary incubation at 56 ± 0.5 °C for 10 min to facilitate the enzymatic digestion of cell walls, followed by 95 ± 0.5 °C heat shock for 5 min to denature nucleases and release genomic DNA. Cellular debris was precipitated by centrifugation at 17,900× g for 5 min at 4 °C. The resulting supernatant containing crude DNA extracts was carefully aspirated using a wide-bore pipette tip (avoiding pellet disturbance) and transferred to a nuclease-free microcentrifuge tube (supplied with the kit) for immediate downstream PCR applications. The ITS sequences of these fungal strains are listed in Table S3.

Apple twigs (Malus domestica Borkh. cv. “Fuji”) were collected from the Apple Experiment Station of the Horticulture College at Ludong University, Yantai County, Shandong Province, China (121.365584° E, 37.528498° N).

2.2. Isolation of the GW2A Strain

Leaf samples from sunflowers, consisting of small sections (5 mm × 5 mm) that included both diseased and healthy tissues, were carefully collected from the Penglai district of Yantai City, Shandong Province, China.

The surfaces were disinfected with 75% ethanol for 30 s, then rinsed three times with sterilized water. Next, the tissues were treated with 10% NaClO for 3 min and rinsed with sterilized ultrapure water. Finally, the sterilized tissues were plated on potato dextrose agar (PDA) (Hangzhou Microbial Reagent Co., Ltd., Yuhang District, Hangzhou, China) and incubated at 25 °C in the dark for one week.

2.3. Identification of the Fungi Strain

Identification of the fungal strain was performed through phylogenetic tree analysis and morphological observation. The fungus was cultivated on PDA medium for 5 days, and its color was observed. The mycelium and spore morphology were observed using confocal microscopy with transmitted light (LSM800, Zeiss, Huangpu District, Shanghai, China).

The fungi genomic DNA was isolated utilizing the DNALyse Amplification Kit (CW0556S, CWBIO, Beijing, China). Following a 5-day cultivation on potato dextrose agar (PDA) at 28 °C, fungal mycelia were aseptically harvested using a sterile surgical blade and transferred into a 1.5 mL sterile microcentrifuge tube. Subsequently, 200 μL of preheated lysis buffer (DNALyse Amplification Kit, CWBIO, Changping District, Beijing, China) was added to the biomass. The mixture underwent sequential thermal treatments: primary incubation at 56 ± 0.5 °C for 10 min to facilitate the enzymatic digestion of cell walls, followed by 95 ± 0.5 °C heat shock for 5 min to denature nucleases and release genomic DNA. Cellular debris was precipitated by centrifugation at 17,900× g for 5 min at 4 °C. The resulting supernatant containing crude DNA extracts was carefully aspirated using a wide-bore pipette tip (avoiding pellet disturbance) and transferred to a nuclease-free microcentrifuge tube (supplied with the kit) for immediate downstream PCR applications. PCR was performed using primers specific to the Alternaria fungus (Table S1). The PCR amplification procedure was as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s, and extension at 72 °C for 60 s. A final extension step was performed at 72 °C for 5 min. Then, the PCR products were sequenced by Beijing Tsingke Biotech Co., Ltd. (Qingdao, China). Phylogenetic trees were generated using the sequences of 28S rRNA, Alt a1, and ITS (Table S1) from the Alternaria fungus with MEGA (7.0.14) software via 1000 bootstrapping replicates and the Neighbor-Joining (NJ) method.

2.4. Determination of the Biological Characteristics of the GW2A Strain

2.4.1. The Effect of Different Carbon Sources on the Growth of the GW2A Strain

The carbon source in the basic culture medium (composed of 0.050% D-(+)-glucose, 0.168% ammonium sulfate, 0.050% potassium dihydrogen phosphate, 0.150% disodium hydrogen phosphate, 0.100% sodium chloride, 0.020% magnesium sulfate heptahydrate, and 2% agar) was altered by replacing D-(+)-glucose with L-arabinose, soluble starch, xylose, sucrose, L-rhamnose, mannitol, sorbitol, or fructose to prepare a basic culture medium containing equivalent masses of the replacement carbon sources. GW2A mycelial blocks with a diameter of 7 mm were inoculated into the center of the carbon source-replaced basic culture medium plates and incubated in a dark incubator at 25 °C for 6 days, after which the colony diameter (mm) was measured. A basic culture medium containing D-(+)-glucose was used as a control. For each condition, three mycelial blocks were inoculated, three plates were used, and the experiments were conducted in triplicate.

2.4.2. The Effect of Different Nitrogen Sources on the Growth of the GW2A Strain

The nitrogen source in the basic culture medium (composed of 0.050% D-(+)-glucose, 0.168% ammonium sulfate, 0.050% potassium dihydrogen phosphate, 0.150% disodium hydrogen phosphate, 0.100% sodium chloride, 0.020% magnesium sulfate heptahydrate, and 2% agar) was altered by substituting it with beef extract, tryptone, ammonium oxalate, sodium nitrate, and urea to prepare a basic culture medium with equivalent amounts of a replacement nitrogen source. Seven-millimeter-diameter GW2A mycelial blocks were inoculated into the center of the nitrogen source-replaced basic culture medium plates, followed by incubation in a dark incubator at 25 °C for 6 days, after which the colony diameter (mm) was measured. A basic culture medium containing ammonium sulfate was used as a control. Three mycelial blocks were inoculated each time, three plates were used, and the experiments were repeated three times.

2.4.3. The Effect of Different pHs on the Growth of GW2A

The pH value of the unsolidified PDA medium was adjusted to 2–10 using 1 M HCl solution and 1 M NaOH solution, respectively, to prepare PDA plates with different pH values. Seven-millimeter-diameter GW2A mycelial blocks were inoculated into the center of the plates, followed by incubation in a dark incubator at 25 °C for 6 days, after which the colony diameter (mm) was measured. Three mycelial blocks were inoculated each time, three plates were used, and the experiments were conducted three times.

2.4.4. The Effect of Different Temperatures on the Growth of GW2A

Seven-millimeter-diameter GW2A mycelial blocks were selected from the edge of activated colonies and inoculated into the center of solid PDA plates. The plates were then placed in incubators set at 10, 18, 25, 28, 37, and 60 °C for dark cultivation. After 6 days, the colony diameter (mm) was measured. Three mycelial blocks were inoculated each time, and the experiments were performed three times.

2.5. Assessment of Antifungal Activity of GW2A

The GW2A strain was co-cultured with several pathogens, including Diaporthe spiculosa, Magnaporthe oryzae, Rhizoctonia solani, Alternaria tenuissima, Colletotrichum gloeosporioides, and Trichothecium roseum. Specifically, GW2A mycelial blocks were placed in the center of the PDA (potato dextrose agar) plate, while mycelial blocks of the other fungi were positioned on both sides. The PDA plates inoculated with fungal cultures were incubated at 25 °C (±0.5 °C) under controlled humidity conditions (65% RH) for a 7-day period. The experiment was conducted three times to verify the reproducibility and reliability of the results.

2.6. The Control Effect of the GW2A Strain on Apple Mold Heart Disease

The control effect of the GW2A strain on apple mold heart disease was evaluated using the method described by He et al., 2024 [37].

Detached four-year-old apple twigs (Malus domestica Borkh. cv. “Fuji”) were cut to approximately 15 cm in length and washed once with distilled water, 75% ethanol, and 2% sodium hypochlorite solution, followed by two rinses with distilled water. Both ends of the twigs were sealed with solid paraffin wax. A wound was then made at the midpoint of each twig using a heated iron nail cap with a diameter of 1 cm. The wound sites were inoculated with 7 mm diameter pieces of GW2A mycelial blocks cultured in PDA for 3 days. For the control group, the wound sites were inoculated with 7 mm diameter pieces of PDA medium blocks. Two days later, the PDA medium blocks were substituted with mycelial blocks of T. roseum of the same dimensions. For the experimental group, the wound site was inoculated with 7 mm diameter GW2A mycelial blocks. Two days later, the GW2A mycelial blocks were replaced with T. roseum mycelial blocks of the same size. GW2A refers to the treatment where apple twigs were initially inoculated with GW2A, followed by inoculation with T. roseum after a two-day interval. PDA indicates the control treatment, where apple twigs were pre-inoculated with PDA medium and then inoculated with T. roseum two days later. All treatments were incubated at 25 °C (±0.5 °C) under controlled humidity conditions (65% RH) for 4 days. The diameter of the lesion was measured starting from the second day after inoculation with T. roseum mycelial blocks. Each treatment was replicated three times, using five twigs per replicate.

Apple fruits during storage (Malus domestica Borkh. cv. “Fuji”) were washed with sterile water, and needle holes approximately 1 mm deep were made at specific positions on the fruit surface using sterile pins. The sites of the needle holes were inoculated with 7 mm diameter GW2A mycelial blocks cultured in PDA for 3 days. For the control group, the wound site was inoculated with 7 mm diameter PDA medium. Two days later, the GW2A mycelial blocks were replaced with T. roseum mycelial blocks of the same size. All treatments were incubated at 25 °C (±0.5 °C) under controlled humidity conditions (65% RH) for 28 days. The lesion diameter was measured at 7, 14, and 28 days after inoculation with T. roseum mycelial blocks. GW2A indicates that apple fruits were pre-inoculated with GW2A and then inoculated with T. roseum two days later. PDA indicates that apple twigs were pre-inoculated with PDA and then inoculated with T. roseum two days later. The experiments were repeated three times with five apple fruits per treatment.

2.7. Analysis of Defense-Related Gene Expression in Apple Trees

The GW2A mycelial blocks were inoculated onto apple twigs for 48 h. Subsequently, the mycelial blocks at the inoculation site were scraped off, and RNA was extracted from the bark at the inoculation site. PDA was inoculated as a control instead of GW2A. For the extraction of total RNA, the FastPure Universal Plant Total RNA Isolation Kit was used (Vazyme, Nanjing, China). For cDNA synthesis, the HiScript II Q RT SuperMix for qPCR was employed (Vazyme, Nanjing, China). Quantitative real-time reverse transcription PCR (qRT-PCR) was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The reference gene EF1α from apples was used as an internal control [37]. The relative expression levels of the tested genes were calculated using the 2^−ΔΔCT method. The experiments were repeated three times.

2.8. Transcriptomic Analysis of T. roseum Treated with GW2A

The T. roseum mycelia treated with PDA and GW2A were collected, with each treatment repeated three times. All samples were sent to OE Biotech, Inc. (Shanghai, China) for transcriptome sequencing analysis.

The libraries were sequenced on an Illumina Novaseq 6000 platform, generating 150 base pair paired-end reads. Each sample produced approximately 46.76 raw reads. The raw reads of fastq format were firstly processed using fastp [38], during which low-quality reads were removed to obtain clean reads. Subsequently, each sample retained about 46.50 million clean reads for further analysis. The clean reads were mapped to the T. roseum reference genome (GenBank ID: GCA_026184415.1) using HISAT2 [39]. The read counts for each gene were determined using HTSeq-count [40]. A differential expression analysis was conducted using DESeq2 (v 3.2.0) [41]. A Q value <0.05 and fold change >2 were set as the threshold for significant differential expression genes (DEGs).

Subsequently, GO [42] and KEGG [43] pathway enrichment analyses were performed on the DEGs based on the hypergeometric distribution algorithm to screen for significantly enriched functional items.

A bioinformatic analysis was performed using the OECloud tools at the OE Biotech Cloud Platform (https://cloud.oebiotech.com/task/, accessed on 13 December 2024). A volcano map (and other graphics) was generated using R v 3.2.0 (https://www.r-project.org/, accessed on 13 December 2024) on the OE Biotech Cloud Platform (https://cloud.oebiotech.com/task/, accessed on 13 December 2024).

2.9. Transcriptome Data

The raw data supporting the article are available online at https://bigd.big.ac.cn/gsa/browse/CRA021665, accessed on 31 December 2024.

2.10. Statistical Analyses

Statistical analysis was performed using DPS software (v19.05), employing an analysis of variance (ANOVA) and significance testing to evaluate data from completely randomized single-factor experiments.

2.11. Experiment Date

All experiments were completed prior to submission. The isolation and identification of the GW2A strain were accomplished in March 2024. The biological characterization of the GW2A strain was completed in June 2024. The antifungal spectrum analysis of the GW2A strain was conducted in July 2024. The in vitro control efficacy of the GW2A strain against apple mold heart disease was evaluated in September 2024. The analysis of immune responses induced by GW2A in apples was completed in October 2024. The transcriptomic analysis of T. roseum treated with GW2A was finalized in December 2024.

3. Results

3.1. Isolation and Identification of Sunflower Fungus

The pathogenic fungus A. angustiovoidea GW2A was isolated from the diseased spots on sunflower leaves (Figure 1A). The GW2A colonies initially appeared white and eventually transformed into a brownish-green color on PDA. The conidiophores, which grow at the ends of the hyphae, are either upright or curved, single or branched, and have diaphragms. Their lengths vary between 18.3 and 54.2 μm, and the widths range from 3.2 to 4.8 μm. The conidia are typically ovoid to obclavate, measuring approximately 28.7–50.4 μm in length and 8.4–24.3 μm in width. They are brown and have 0–2 longitudinal septa and 1–4 transverse septa. Some conidia have short beaks at their ends (Figure 1B,C).

Based on the morphological characteristics, it could be determined that the fungus belonged to Alternaria sp. [44,45]. In order to further identify and classify it, the genomic DNA was extracted from a 7-day-old culture. The internal transcribed spacer (ITS), 28S ribosomal RNA (28S rRNA), and Alternaria major allergen (Alt a1) were amplified with primer ITS1/ITS4 [46], 5.8S-F/28S-R (Table S1), and Alt-f/Alt-r [47], respectively.

A BLASTn analysis of 28S rRNA (28S rRNA: PQ276929), Alt a1(Alt a1:PQ303561), and ITS (ITS: PQ278795) sequences failed to provide a conclusion about the species of GW2A. So a phylogenetic tree of Alternaria fungi was constructed by concatenating 28S rRNA, Alt a1, and ITS gene sequences. The phylogenetic tree showed that the nearest relatives to GW2A was A. angustiovoidea CBS 195.86 (Figure 2).

3.2. Biological Characteristics of GW2A

3.2.1. Effects of Carbon Sources on Mycelial Growth

The GW2A strain can grow in culture media containing various carbon sources. The hyphae of A. angustiovoidea grow most rapidly when fructose, soluble starch, D-(+)-glucose, and xylose are used as carbon sources, with no significant difference in growth rate between these three carbon sources. In contrast, the fungus growth rate is slower when xylose and mannitol are used compared to sorbitol, sucrose, and arabinose. The slowest fungus growth rate is observed when rhamnose is the sole carbon source (Figure 3A). This suggests that fructose, soluble starch, D-(+)-glucose, and xylose are suitable carbon sources for the growth of the GW2A strain.

3.2.2. Effects of Nitrogen Source on Mycelial Growth

The GW2A strain can grow in media containing various nitrogen sources. The mycelium of GW2A grows most rapidly when ammonium sulfate is used as the nitrogen source, resulting in a significantly larger colony diameter (mm) compared to other nitrogen sources, followed by beef extract, sodium nitrate, and tryptone. In contrast, the fungus grows slowest when ammonium oxalate is used as the nitrogen source, followed by urea (Figure 3B). This indicates that ammonium sulfate is a more suitable nitrogen source for the growth of the GW2A strain.

3.2.3. Effects of pH on Mycelial Growth

The GW2A strain can grow at pH values ranging from 2 to 10. Mycelium grows slowest under pH 3 conditions. The growth rate of the fungus accelerates with the increase in pH value between pH 2 and 4. Subsequently, as the pH increases further, the growth rate of the fungus remains relatively stable (Figure 3C). These results indicate that the GW2A strain has strong adaptability to pH changes.

3.2.4. Effects of Temperature on Fungal Growth

The GW2A strain can grow at temperatures ranging from 18 to 37 °C. The highest growth rate is reached at 25 °C and 28 °C. Then, the growth rate declines with an increase in temperature. Thereafter, the growth rate continues to decline and ceases at 60 °C (Figure 3D). This demonstrates that 25 °C and 28 °C are the optimal growth temperatures for the strain GW2A.

3.3. The GW2A Strain Can Restrict the Growth of T. roseum

Although Alternaria is primarily a pathogenic fungus, it also has the potential to serve as a biocontrol agent. To investigate the inhibitory effects of GW2A on various types of pathogenic fungi, with the goal of understanding the specific inhibitory effect of GW2A, we assessed its inhibitory effect on the fungus growth of Diaporthe spiculosa, Magnaporthe oryzae, Rhizoctonia solani, A. tenuissima, Colletotrichum gloeosporioides, and T. roseum (Figure 4A). The results showed that GW2A had no inhibitory activity against most fungi but had a slight inhibitory effect on T. roseum (Figure 4B). The GW2A strain can restrict the growth of T. roseum.

3.4. The Prevention Effect of GW2A on T. roseum in Apple Twigs and Fruits

To investigate whether the GW2A strain could inhibit T. roseum infection, we used apple twigs and fruits to assess its effects. The results showed that the lesion diameter on the twigs pre-inoculated with GW2A was smaller than that of the control samples at 2, 3, and 4 days post-inoculation with T. roseum (Figure 5A). A statistical analysis revealed that the lesion diameter was significantly reduced by 83.9 (±1.3%) at 3 days and 92.6 (±2.8%) at 4 days post-inoculation, respectively. (Figure 5B).

Similarly, the lesion diameter on the apple fruits was smaller than that of the control samples at 7, 14, and 28 days post-inoculation with T. roseum (Figure 6A), with reductions of 58.2%, 46.5%, and 29.7%, respectively (Figure 6B).

3.5. The Immune-Inducing Effect of GW2A on Apple Twigs

To investigate whether GW2A induces resistance in apples, we analyzed the expression of seven disease-resistance-related genes (MdPR1, MdPR2, MdPR4, MdPR5, MdNPR1, MdRBOHD, and MdCYP81F2) in apple twigs treated with GW2A for 48 h. The results showed that all the genes were upregulated after the apple twigs were treated with GW2A. The expression of MdNPR1 and MdRBOHD was slightly upregulated after GW2A treatment. Meanwhile, the expression of MdNPR1, MdPR4, and MdCYP81F2 was significantly upregulated, with fold changes of 3.38, 4.16, and 4.45, respectively (Figure 7).

3.6. Transcriptomic Analysis of T. roseum Treated with GW2A

To elucidate the inhibitory mechanism of GW2A on T. roseum, we employed RNA-seq to analyze differentially expressed genes (DEGs) between untreated T. roseum (Tr-ck) and GW2A-treated T. roseum (Tr-bt). A total of six RNA libraries, comprising two samples with three biological replicates each, were analyzed. A total of 347 DEGs (257 upregulated and 90 downregulated) were identified (Figure 8A). When fungal growth is inhibited, physiological and metabolic processes may also be affected. Consequently, we performed GO and KEGG enrichment analyses on pathways associated with the growth and development of T. roseum during GW2A inhibition to elucidate the potential regulatory roles of the DEGs. The upregulated DEGs were found to be enriched in GO terms linked to biological processes and molecular functions, such as transmembrane transport and the tricarboxylic acid cycle process. Additionally, they were over-represented in KEGG pathways, including kinase signaling, ribosome biogenesis, and the metabolism of certain complex compounds in the tricarboxylic acid cycle (Figure 8B,D). The downregulated DEGs were enriched in GO terms related to biological processes and molecular functions, such as polysaccharide transport, metabolism and degradation processes, the defense response to bacteria, and the amine metabolic process. The molecular functions of these downregulated DEGs were enriched in polysaccharide hydrolases, oxidoreductases, and intermediate processes in carbohydrate metabolism (Figure 8C). Meanwhile, the downregulated DEGs were over-represented in KEGG pathways, including carbohydrate metabolism, amino acid metabolism, and antibiotic synthesis (Figure 8E). The results indicated that GW2A inhibited T. roseum growth by suppressing or disrupting carbohydrate metabolism processes, by damaging cellular structures (cell walls and membranes), and by suppressing the function of polysaccharide hydrolases.

4. Discussion

As an environmentally friendly agriculture advancement, biological control is becoming increasingly important in agricultural production. Apple mold heart disease significantly affects the economic value and edibility of various harvested fruits. The disease, caused by a variety of pathogens including Trichothecium, Alternaria, Fusarium, and Pseudomonas, is difficult to detect in its early stages [48]. Wet rot plaques on the apple’s surface become visible only when the core is severely moldy [49,50]. In this study, we successfully isolated the fungal species A. angustiovoidea from sunflower plants, a species which causes leaf spot disease on sunflower leaves.

The genus Alternaria comprises over 250 species, known for their strong ability to utilize various carbon and nitrogen sources [45,51,52]. Our results indicate that fructose, soluble starch, D-(+)-glucose, and xylose are suitable carbon sources for the growth of the GW2A strain. Ammonium sulfate is a more suitable nitrogen source for the GW2A strain. The growth and reproduction of Alternaria species have specific temperature requirements, with an optimal range of 20–30 °C for pathogen growth [45]. In our study, A. angustiovoidea can grow within a temperature range of 18–37 °C, with an optimal growth temperature of 25 °C. Additionally, GW2A exhibits strong adaptability to pH changes, being able to survive under conditions ranging from pH 2 to 10.

As Alternaria spp. have a broad host range in nature, they can cause significant economic losses to the country and farmers. However, some species of Alternaria can be utilized as biocontrol agents against other pathogenic fungi. For instance, A. alternata, isolated from Sclerocarya birrea, can secrete the compound 2-fluorobenzoic acid heptadecyl, which exhibits broad-spectrum activity, particularly against Gram-negative multidrug-resistant (MDR) contemporary pathogens [53]. Similarly, an endophytic Alternaria sp., RL4, can produce bioactive compounds that are effective against Staphylococcus aureus and Listeria monocytogenes [54]. In line with these findings, our results demonstrate that A. angustiovoidea GW2A is an effective resource for controlling apple mold heart disease. Apple mold heart disease typically emerges during the postharvest storage phase, with pathogens being most vulnerable to initial infection prior to flowering, during flowering, and at petal fall [50]. Given this vulnerability, targeted plant protection measures should be implemented at various stages to prevent and control apple mold heart disease. Maintaining orchard hygiene by promptly removing withered branches and fallen leaves is crucial, as it helps minimize pathogen-breeding environments and reduces initial pathogen infections [49,50]. In our study, the lesion diameter on apple twigs decreased by 83.9% after 3 days and 92.6% after 4 days following preemptive treatment with GW2A. The lesion diameter on apple fruits decreased by 58.2% after 7 days, 46.5% after 14 days, and 29.7% after 28 days following preemptive treatment with GW2A. The results demonstrate that GW2A is particularly effective for biological control, achieving optimal effects during the initial infection stage. This is because once the pathogen colonizes the apple fruits, the nutrient-rich environment facilitates the spread and infection by T. roseum, thereby reducing the biocontrol efficacy of GW2A. If T. roseum penetrates the apple’s interior, chemical fungicides struggle to manage the disease effectively.

Most biocontrol fungi exhibit effects against a variety of pathogenic fungi. For example, Arcopilus aureus can strongly inhibit the growth of Colletotrichum spaethianum, A. tenuissima, Nigrospora oryzae, Phytophthora capsici, Fusarium oxysporum, F. moniliforme, Peniophora incarnata, Pestalotiopsis calabae, and F. fujikuroi. Phenylethyl alcohol and 3,5-dihydroxytoluene can inhibit the growth of F. fujikuroi [55]. In contrast, GW2A specifically inhibits the growth of T. roseum and shows no inhibitory effects on other fungi. It is speculated that GW2A may secrete specific compounds that exert inhibitory effects exclusively on T. roseum.

Numerous microbes secrete elicitors that trigger plant disease resistance mechanisms. These mechanisms include oxidative bursts, the accumulation of pathogenesis-related proteins, hypersensitivity responses, and the upregulation of defense-related genes [56,57,58]. Biocontrol microbes, in particular, function to induce plant resistance. For example, T. roseum (T203) modulates the expression of genes involved in the jasmonate/ethylene signaling pathways of induced systemic resistance (ISR), such as Lox1, Pal1, ETR1, and CTR1, thereby inducing resistance in cucumbers [59]. Our study demonstrated that GW2A can induce the upregulation of various defense-related genes, implying that GW2A can inhibit pathogen infection and enhance apple resistance.

Plant pathogens degrade carbohydrates and obtain the necessary monosaccharides for growth and reproduction by secreting cell wall-degrading enzymes [60]. Inhibition of these pathways leads to a significant reduction in metabolic activity, thereby impairing the cellular capacity for carbohydrate biosynthesis required to fulfill nutritional demands. This metabolic disruption ultimately imposes severe constraints on hyphal growth. Transcriptome analysis in our study revealed that GW2A inhibits polysaccharide hydrolases, oxidoreductases, and intermediate steps in the carbohydrate metabolism of T. roseum, thereby restricting its growth. To counteract this inhibition, T. roseum may enhance the transmembrane transport and metabolic processes of its existing nutrients, leading to an enrichment of DEGs related to transmembrane transport and the tricarboxylic acid (TCA) cycle.

The development and utilization of biocontrol fungi represents a sustainable and eco-friendly strategy to reduce pesticide reliance and promote green agriculture [61,62,63,64]. Our strain, GW2A, has demonstrated significant efficacy in inhibiting the harm caused by T. roseum through various mechanisms. Integrating GW2A into agricultural systems is expected to significantly reduce the dependence on chemical pesticides. This reduction will help minimize environmental pollution, preserve biodiversity, and enhance ecosystem sustainability.

While the efficacy of GW2A has been well established in controlled environments, its performance in field conditions can be influenced by a variety of factors, including temperature, humidity, and soil microbiota. Given that GW2A is a pathogen of sunflower leaves, its application as a biocontrol agent for apple mold heart disease is not feasible in regions where apple orchards and sunflower fields are in close proximity. Therefore, its safety and potential ecological risks must be rigorously evaluated. Potential concerns include unintended effects on non-target organisms, genetic stability, and long-term ecological impacts. Comprehensive risk assessments, encompassing both laboratory and field studies, are essential to ensure its safe integration into agricultural systems. Robust regulatory frameworks and monitoring protocols should be established to mitigate potential risks, while maximizing the benefits of GW2A for crop protection and environmental health. To facilitate the wider application of GW2A in agricultural production, further research is needed to explore its environmental stability, optimize its application formulations, and evaluate its efficacy against other pathogens.

5. Conclusions

This study highlights the potential of the Alternaria angustiovoidea strain GW2A as a dual-purpose microbial agent, capable of both causing sunflower leaf diseases and serving as an effective biocontrol agent against apple mold heart disease. These findings are particularly significant in the context of global agricultural trends, which increasingly emphasize sustainable farming practices and a reduced reliance on chemical pesticides. The specific inhibitory effects of GW2A on Trichothecium roseum, its ability to prevent the spread of apple mold heart disease, its disruption of carbohydrate metabolism in T. roseum, and its capacity to induce resistance in apples collectively underscore its potential as a sustainable alternative to synthetic fungicides. However, the current study has limitations, including the need for field trials to validate these findings under real-world conditions and for a deeper exploration of the mechanisms underlying GW2A’s biocontrol efficacy. Future research should also investigate the potential ecological impacts of deploying GW2A on a larger scale, including its interactions with non-target organisms and long-term effects on soil health. Addressing these gaps will be crucial for advancing the development of GW2A as a viable biocontrol agent and aligning it with the global shift toward environmentally responsible agricultural practices.