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Article

Pathogenic Factors and Mechanisms of the Alternaria Leaf Spot Pathogen in Apple

by
Chen Cao
,
Shun Gong
,
Yan Li
,
Jinqi Tang
,
Tianzhong Li
and
Qiulei Zhang
*
Laboratory of Fruit Cell and Molecular Breeding, China Agricultural University, Beijing 100193, China
*
Author to whom correspondence should be addressed.
Horticulturae 2024, 10(3), 212; https://doi.org/10.3390/horticulturae10030212
Submission received: 8 October 2023 / Revised: 4 January 2024 / Accepted: 7 January 2024 / Published: 23 February 2024
(This article belongs to the Section Plant Pathology and Disease Management (PPDM))
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Abstract

:
Alternaria leaf spot seriously threatens the sustainable development of the global apple industry, causing significant losses and reducing fruit quality and yield. The causal agent Alternaria alternata f. sp. mali (Alternaria mali, ALT) produces various molecules to modulate infection, such as cell wall-degrading enzymes, toxins, and elicitor-like molecules. ALT produces the host-specific AM-toxin, an important pathogenicity factor. ALT also releases effectors into apple cells that modify host defense, but these proteins have not yet been described. Here, we identified the pathogenic fungal types responsible for early defoliation from diseased leaves of Fuji (Malus domestica cv. ‘Fuji’) apple collected from five districts in Shandong Province, China. The ALT isolates ALT2 to ALT7 were pathogenic to four apple cultivars, with ALT7 being the most aggressive. We extracted mycotoxins (AM-toxin-2 to AM-toxin-7) from each isolate and used them to treat different apple varieties, which led to leaf-spot symptoms and damaged chloroplasts and nuclear membranes, followed by cell death. AM-toxin-7 produced the most severe symptoms, but chloroplasts remained intact when the mycotoxin was inactivated. Mass spectrometry identified 134 secretory proteins in ALT7 exosomes, and three secreted proteins (AltABC, AltAO, and AltPDE) were confirmed to be involved in apple pathogenesis. Therefore, ALT secretes AM-toxin and secretory proteins as an infection strategy to promote fungal invasion and overcome the host defense system.

1. Introduction

Apple (Malus × domestica) is one of the most important fruit crops worldwide. Apple production and cultivation are widely practiced throughout China, with the largest apple production areas concentrated in Shandong and Shanxi provinces, accounting for 89.1% of the cultivation area and 84.2% of yield in China. However, the productivity of the apple industry in China is constrained by several fungal diseases [1,2,3]. Alternaria leaf spot (Alternaria alternata apple pathotype) caused by Alternaria alternata f. sp. mali (Alternaria mali, ALT) [4,5] is an economically important fungal disease of apple that limits the economic productivity of the apple industry and threatens apple production in many areas worldwide [4,6,7,8]. In China, Alternaria leaf spot of apple was first reported in the 1970s and is currently considered to be among the four most serious diseases in all major apple production areas of the country [9,10,11].
In general, when fungi attack apple leaves, they produce toxins and effectors, leading to the accumulation of H2O2 and eventual cell death [4,12]. In addition, ALT produces the host-specific AM-toxin and effectors, which disrupt plant metabolism [2,3] and increase the incidence of defoliation and fruit drop, leading to a decline in fruit quality and production [13,14,15]. ALT infects apple leaves in a complex manner. ALT produces a mycotoxin that plays important roles in fungal colonization and disease induction; its action sites include the plasma membrane and chloroplasts [16].
Necrotic lesions that develop due to treatment with mycotoxins are similar to those produced by the pathogen, highlighting the crucial roles of these toxins in plant pathogenesis [17,18]. The AM-toxin known to be secreted by ALT is a cyclic peptide. Successful infection of the host plant induces cell membrane damage (disrupted cell wall proteins), the production of reactive oxygen species (ROS), and increased H2O2 accumulation, followed by cell death [14,19]. The AM-toxin initially alters the fluidity of the plasma membrane of the host cell. The plasma membrane abnormally invaginates into the cytoplasm, the cell wall degrades, the chloroplast structures are damaged, and the cell dies [20,21,22,23].
Plants have evolved a primary defense system that responds to pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors, a signaling cascade that is termed PAMP-triggered immunity (PTI) [24,25]. PTI causes rapid defense reactions, such as the production of ROS, ion fluxes, and cell wall reinforcement. However, successful pathogens interfere with PTI by delivering effectors, evading recognition, or suppressing signaling pathways. In turn, the host uses resistance proteins to recognize the effectors, leading to the secondary defense system of effector-triggered immunity (ETI) [24,25]. In addition to AM-toxin, ALT also releases a set of effectors into apple cells that modify the host defense reaction. However, the effector proteins of ALT have not been identified.
Whereas toxins and elicitor-like molecules have been the focus of most studies of this disease, our goal was to characterize morphological and pathogenic diversity among isolates of Alternaria collected from apples in the Yantai apple-growing region of China, to examine the role of toxic metabolites and to identify virulence factors of ALT on apple. In this study, we collected diseased leaves of Fuji apple from five districts of Shandong Province, China, representing the types of pathogenic fungi that lead to early defoliation in the apple-growing region of Yantai via tissue isolation, morphological observations, and molecular identification. We obtained six purified isolates of ALT, named ALT2 to ALT7. After their mycotoxins were extracted, we treated different apple varieties with compounds derived from each isolate, which elicited similar phenotypes to those induced by ALT. The mycotoxins disrupted the plasma membrane, but when we inactivated the mycotoxins, the chloroplasts remained intact. Liquid chromatography–mass spectrometry (LC–MS) identified 134 secretary proteins that might modulate ALT infection. To investigate the pathogenic mechanism of these secreted proteins, we identified three secreted proteins: AltABC, AltAO, and AltPDE, overexpressed these proteins in ‘Hanfu’ apple leaves, and observed a decrease in disease resistance of transgenic Hanfu after inoculation with ALT7. Our findings provide a theoretical basis for developing effective disease prevention and control measures for Alternaria leaf spot of apple.

2. Materials and Methods

2.1. Plant materials and Growth Conditions

From July to September 2021, diseased apple (cv. ‘Fuji’) leaves were collected from five districts in Yantai, Shandong Province: Laishan District, Muping District, Haiyang City, Qixia City, and Zhaoyuan City. Two sampling sites were set up in each district (orchard management levels were basically the same), and 10 leaves (middle and lower layers of fruit trees) were collected from each sampling site. After leaf samples were picked, they were sealed in fresh bags and numbered, stored on ice, and brought back to the laboratory for the immediate isolation of pathogenic fungi [8].
‘Golden Delicious’, ‘Gala-3’, ‘Shangdong Gala’, and ‘Hanfu’ apple plants were grown by tissue culture on Murashige and Skoog (MS) medium containing 1 mg L−1 6-benzylaminopurine, 0.5 mg L−1 3-indoleacetic acid, and 2 mg L−1 gibberellin A3 in a climate-controlled culture room at 25 ± 1 °C under fluorescent lights at ~100 μmol m−2 s−1 in a 16 h photoperiod (Philips Lighting TL5 28W/865). The plants were transferred to fresh medium every 4 weeks. Approximately 4-week-old apple seedlings were used in the fungal infection and toxin injection experiments when the fifth leaf of the seedling had expanded (leaf length: 2 cm) [25].

2.2. Isolation, Culture, and Identification of Pathogenic Fungi

The conventional tissue isolation method was used for the isolation of pathogenic fungi. The diseased leaves were cleaned, and the tissue at the diseased–healthy leaf junction was collected, cut into 5 mm × 5 mm pieces, disinfected with 75% alcohol for 30 s, followed by 1.5% NaClO3 for 3 min, and rinsed 3–5 times with sterile water. The disinfected leaf pieces were gently placed on potato dextrose solid medium (PDA) containing streptomycin, using 3–5 leaf pieces per dish, and incubated in the dark at a constant temperature of 28 °C.
The fungi were cultured on PDA medium for 5–7 days, and external characteristics such as the size, color, and shape of the top and bottom of each colony were observed and recorded. A fully grown single colony was picked with a dissecting needle, placed on a slide in a drop of sterile water, and covered with a coverslip. The color, size, shape, and other characteristics of the spores and mycelium were observed under a microscope [11].
DNA was extracted from the fungi using a modified cetyltrimethylammonium bromide method, and the DNA concentration was measured using an ND-1000 NanoDrop spectrophotometer (Thermo Fisher Scientific, San Jose, CA, USA). Primers for plant pathogenic Marssonina coronaria, Alternaria alternata f. sp. mali, and Colletotrichum spp. were synthesized by Biotech Bioengineering (Supplemental Table S1). DNA from the strains was extracted from diseased leaf pieces, amplified by PCR, and detected by 1.5% agarose gel electrophoresis.
The forward primer for ITS sequence amplification was ITS1 (5′-TCCGTAGGTGAACCTGCG-3′), and the reverse primer was ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The primers for β-tubulin sequence amplification were TUB2-T1-F (5′-AACATGCGTGAGATTGTAAGT-3′) and TUB2-Bt2b-R (5′-ACCCTCAG TG TAG TGACCCTTGGC3′). The primers for Alt a 1 sequence amplification were ALTa1-f (5′-CTCTCTCTTCGCCGCCGCTGG-3′) and ALTa1-r (5′-GGCAACACCCTGGCAGACAAAGT-3′). The PCR reaction system of 25 μL included: 1.5 μL of DNA template, 0.75 μL of each 10 μmol·L-1 primer, 12.5 μL of 2 × Taq PCR MasterMix, and 9.5 μL of ddH2O. The ITS sequence amplification program was: 94 °C for 3 min; 94 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min, 35 cycles; 72 °C for 2 min. The β-tubulin sequence amplification program was: 94 °C for 3 min; 94 °C for 30 s, 58 °C for 30 s, 72 °C for 45 s, 35 cycles; 72 °C for 2 min. The Alt a 1 sequence amplification program was: 94 °C for 3 min; 94 °C for 30 s, 61 °C for 30 s, 72 °C for 1 min, 35 cycles; 72 °C for 2 min. The PCR products were sequenced in both directions by Sangon Biotech Co., Ltd., Shanghai, China.

2.3. Toxin Extraction

The test strains ALT2/3/4/5/6/7 were incubated on PDA medium for 5–7 d. The colonies were punched into fungal discs with a 0.6 cm diameter puncher, inoculated into potato dextrose broth (PDB), and incubated for 22 d at 24 °C in the dark. The culture was filtered through four layers of gauze, centrifuged at 5000 r/min for 15 min, passed through a vacuum filter, and quantitatively concentrated in a constant temperature water bath at 95 °C. The concentrated culture filtrate was combined with an equal volume of ethyl acetate in a partition funnel and shaken for 10–15 min. The solution was left to stand for 30 min and divided into two layers: the upper layer was removed, and the lower layer was extracted two more times. The upper layer was collected three times, and the ethyl acetate was removed by evaporation under reduced pressure in a constant temperature water bath at 60 °C. The solution was diluted to the original volume with sterile water and stored at 4 °C.

2.4. Toxin Inactivation

The effect of toxin inactivation was determined on apple seedling leaves. A certain concentration of the toxin of ALT2/3/4/5/6/7 was mixed with an equal volume of KMnO4 and shaken for 2 h. Gala-3 and Golden Delicious seedling leaves were punctured with a sterile needle, and an equal volume of the toxin mixture was inoculated on the punctured leaves. Three biological replicates were performed, each with approximately 25 leaves.

2.5. Fungal and Toxin Inoculation Assay

Four-week-old in vitro-grown seedlings were used for inoculation, which was performed as described [8]. A 2 × 105 cfu/mL spore suspension or the toxin of ALT2/3/4/5/6/7 was injected into two regions on the abaxial side of each leaf of Gala-3 and Golden Delicious, avoiding the major veins. The seedlings were incubated in the dark. At 48 h post-inoculation, lesion areas were measured using Image J 1.53e, and lesion areas (%: area of diseased leaf/total area per leaf) were calculated. Three biological replicates were performed, each with approximately 25 leaves.

2.6. Confocal Microscopy

Propidium iodide (PI) was used to stain the nuclei in cells, and PI and chlorophyll fluorescence were visualized under a confocal laser scanning microscope (Olympus FV3000, Tokyo, Japan). The nuclei were observed at excitation light wavelengths of 580–610 nm and chlorophyll at 629–670 nm [26].

2.7. LC-MS Analysis to Identify Secreted Proteins

Young mycelium and spores of ALT7 were inoculated into 100 mL of liquid PDB medium and incubated at 28 °C, 180 rpm. After 20 days, spores and mycelia were removed with a 0.22 μm filter membrane, and the culture medium was concentrated using an Amicon Ultra-15 centrifugal filter to obtain a culture filtrate of ALT7 for LC-MS analysis.
After adjusting the pH to 8.5 with 1 M ammonium bicarbonate, total protein (100 μg) extracted from each sample was chemically reduced for 1 h at 60 °C by adding 10 mM DTT and carboxyamidomethylated in 55 mM iodoacetamide and incubating for 45 min at room temperature in the dark. Trypsin Gold (Promega, Madison, WI, USA) was added to a final substrate/enzyme ratio of 30:1 (w/w). The trypsin digestion mixture was incubated at 37 °C for 16 h. After digestion, the peptide mixture was acidified with 10 μL formic acid (FA) and subjected to MS analysis [27,28].
Following protein digestion, each peptide sample was desalted through a Strata X column (Phenomenex, CA, USA), dried under a vacuum, and resuspended in 200 μL of buffer A (2% acetonitrile (ACN), 0.1% FA). After centrifugation at 4 °C, 20,000× g for 10 min, the supernatant was recovered to obtain a peptide solution at a final concentration of approximately 0.5 μg. A 10 μL aliquot of supernatant was loaded onto the 2 cm C18 trap column of an LC-20AD nanoHPLC system (Shimadzu, Kyoto, Japan) using an autosampler. The peptides were eluted on a 10 cm analytical C18 column (inner diameter 75 μm). The samples were loaded at 8 nl/min for 4 min, and a 44 min gradient was run at 300 nl/min, starting with 2–80% B (98% ACN, 0.1% FA), followed by a 2 min linear gradient to 80%, holding at 80% B for 4 min, and a return to 5% over the course of 1 min. The peptides were subjected to nanoelectrospray ionization followed by tandem mass spectrometry (MS/MS) in a Q Exactive system (Thermo Fisher Scientific, San Jose, CA, USA) coupled online with HPLC [29,30].
Isolated peptides were compared to the ALT genome (GCF_001642055.1), and proteins that exceeded the identification threshold were selected for secreted protein analysis. Protein sequences containing signal peptides were screened using SignalP (https://services.healthtech.dtu.dk/services/SignalP-6.0/, 22 April 2023). TMHMM (https://services.healthtech.dtu.dk/services/TMHMM-2.0/, 14 June 2023) was used to find proteins that do not contain transmembrane structural domains that are assumed to be secreted proteins. The top ten of these with the highest LC–MS analysis scores were selected.

2.8. Overexpression of Three ALT7 Secretory Proteins

For overexpression experiments, the full-length sequences of three selected secretory proteins, AltABC, AltAO, and AltPDE, were cloned into the pFGC5941 vector (GenBank AY310901) using NcoI/BamHI restriction sites. The empty pFGC5941 vector was used as a control [8,25]. The vectors were transformed into GV3101, an Agrobacterium tumefaciens strain, using the heat shock transformation method. The cloning primers are listed in Table S1. Four-week-old Hanfu seedling leaves were infiltrated with an overexpression construct of A. tumefaciens. Following agroinfiltration, the infiltrated seedlings were transferred to a fresh MS culture medium for 4 days to prevent wilting of the apple plantlets, at which time the lesion area was quantified and sampled for fungal biomass using the primers listed in Table S1.

2.9. RT-qPCR

Three different RT-qPCR experiments were conducted:
(1)
The relative expression of toxin genes AMT1 and AMT4 was assessed in ALT7-inoculated Gala-3, Golden Delicious, Shandong Gala, and Hanfu (Section 2.5).
(2)
The relative expression of secretory proteins AltABC, AltAO, and AltPDE was assessed in transgenic OE-AltABC-Hanfu, OE-AltAO-Hanfu, and OE-AltPDE-Hanfu, respectively (Section 2.9).
(3)
Fungal biomass was quantified in transgenic OE-AltABC-Hanfu, OE-AltAO-Hanfu, and OE-Alt-PDE-Hanfu seedlings after inoculation with ALT7 (Section 2.9).
(4)
Total RNA was extracted from apple leaves using an EASY Spin Kit (Biomed Biotechnology Co., Ltd., Beijing, China), amplified using oligo-dT primers (Takara Biomedical Technology Co., Ltd., Beijing, China), and reverse-transcribed into cDNA (see Table S1 for primers). Real-time PCR (RT-PCR) was performed using SuperReal PreMix Plus (SYBR Green) (Tiangen, FP205, Beijing, China) under the following cycling conditions: 40 cycles of 95 °C for 10 sec and 60 °C for 30 s (Applied Biosystems 7500). Relative RNA abundance was calculated using the 2−∆∆Ct method [28], with MdActin (NCBI XM_008365636.2) as the reference gene.

3. Results

3.1. Collection of Apple Leaf Spot Pathogen Strains

To identify the pathogens causing leaf spot and defoliation of Fuji apple leaves in the Yantai apple production area, we selected leaves with typical symptoms from orchards in the Laishan District, Muping District, Haiyang City, Qixia City, and Zhaoyuan City (Figure 1 and Supplemental Figure S1). We identified symptoms and pathogen morphology for 124 diseased leaf samples, compared their morphological characteristics, and performed molecular identification. The incidence of Alternaria leaf spot was the highest, which was the main disease in this region, followed by Marssonina leaf spot (MLS), while the incidence of Glomerella leaf spot (GLS) was the lowest, with disease incidence of 69.5%, 35.3%, and 6.8%, respectively (Table 1). Whereas many studies of apple leaf-spot diseases have been performed, few studies have focused on the pathogenicity of Alternaria leaf spot. We, therefore, studied this pathogen specifically to help prevent and control apple fungal diseases affecting the apple industry in China.

3.2. Morphological Characterization and Sequence Analysis of Single-Spore Isolates

We single-spored these 124 samples and obtained purified strains with a focus on Alternaria species. Preliminary microscopy examination and molecular identification revealed that the pathogen was ALT (Figure 2). We obtained six ALT isolate strains, named ALT2, ALT3, ALT4, ALT5, ALT6, and ALT7. The colonies appeared light gray to dark gray. We confirmed their identities by PCR using ITS, beta-tubulin, and alt a1 primers (Figure 2B,D). ITS, Beta-tubulin, and Alt a1 sequencing comparisons showed that ALT2-ALT7 were all A. alternata strains (Table 2). ALT2 sequencing comparisons were ITS 98.95%, Beta-tubulin 99.42%, and Alt a1 99.54%. ALT3 sequencing comparisons were ITS 99.63%, Beta-tubulin 99.42%, and Alt a1 98.44%. ALT4 sequencing comparisons were ITS 99.81%, Beta-tubulin 99.42%, and Alt a1 99.54%. ALT5 sequencing comparisons were ITS 99.82%, Beta-tubulin 100%, and Alt a1 100%. ALT6 sequencing comparisons were ITS 99.12%, Beta-tubulin 98.83%, and Alt a1 98.63%. ALT7 sequencing comparisons were ITS 99.47%, Beta-tubulin 99.22%, and Alt a1 98.13%.

3.3. Pathogenicity Assays of ALT2 to ALT7 on Apple

To verify that the ALT2 to ALT7 strains exhibit pathogenicity in apple, we inoculated four varieties of apple with spore suspensions and observed disease symptoms after inoculation.
Forty-eight hours after inoculation with an individual isolate (ALT2 to ALT7) on apple cultivar ‘Gala-3’, lesions developed in the leaves (Figure 3A). The average lesion area (measured as a percentage of total leaf area) was significantly higher in ALT7-inoculated Gala-3 seedlings than in seedlings inoculated with any of the other strains, demonstrating that ALT2 to ALT7 are pathogenic to apple and that ALT7 was the most aggressive (Figure 3B).
In addition to Gala-3, we tested the susceptible apple cultivar ‘Golden Delicious’. Following individual inoculation with ALT2 to ALT7, the relative lesion areas in Golden Delicious were 24.5% (ALT2), 26.7% (ALT3), 27.4% (ALT4), 30.1% (ALT5), 21.4% (ALT6), and 36.1% (ALT7), indicating that ALT7 produced the most severe leaf-spot symptoms (Figure 3C,D). Similarly, when we inoculated apple cultivar ‘Shandong Gala’ with ALT2 to ALT7 individually, symptoms appeared (necrosis) on each leaf, demonstrating that ALT2 to ALT7 are pathogenic to this apple cultivar as well (Figure 3E,F).
We also tested the resistant variety ‘Hanfu’. Individual inoculation with ALT2 to ALT7 caused necrosis in Hanfu seedlings at 48 h after treatment (Figure 3G). The average percentage of lesion area in ALT7-inoculated Hanfu seedlings (31.1%) was significantly higher than that of Hanfu seedlings inoculated with any of the other strains (9.25% (ALT2), 10.5% (ALT3), 15.0% (ALT4), 18.8% (ALT5), and 9.9% (ALT6)), demonstrating that ALT2 to ALT7 are pathogenic to apple and that ALT7 was the most aggressive (Figure 3H).
To investigate the growth of hyphae and spores in ALT7-inoculated apple leaves from 0 to 48 h after treatment, we stained ALT7-inoculated apple leaves with aniline blue to detect fungal hyphae at 0, 24, or 48 h after inoculation (hpi). At 24 hpi, the hyphae could be observed in ALT-inoculated Gala-3 (Supplemental Figure S2A), Golden Delicious (Supplemental Figure S2B), and Shandong Gala (Supplemental Figure S2C) leaves. At 48 hpi, mycelial growth was more vigorous in the three susceptible cultivars compared with the resistant cultivar (Supplemental Figure S2A–C). Indeed, we observed few hyphae in the leaves of the resistant cultivar Hanfu at 24 hpi. At 48 hpi, mycelial growth was more vigorous in Hanfu (Supplemental Figure S2D).
The ALT genes AMT1 and AMT4 are both required for AM-toxin biosynthesis. We, therefore, measured the expression of AMT1 and AMT4 in various apple seedlings inoculated with strains ALT2/3/4/5/6/7 (Figure 4). AMT1 and AMT4 were expressed at higher levels in ALT7-inoculated Gala-3, Golden Delicious, Shandong Gala, and Hanfu seedlings compared to the control. The expression patterns of AMT1 and AMT4 were basically consistent with the phenotypes of inoculated apple seedlings (Figure 4).

3.4. Phenotypes and Effects of AM-Toxins on Apple Leaves

To further study the pathogenesis of ALT, we extracted mycotoxins from ALT2 to ALT7. We named these toxins AM-toxin-2, AM-toxin-3, AM-toxin-4, AM-toxin-5, AM-toxin-6, and AM-toxin-7, respectively (Figure 5A). We treated Gala-3 leaves with each toxin individually. At 48 h after treatment, we observed significant changes in the toxin-treated plants, with necrosis appearing on all leaves except control (uninoculated Gala-3) seedlings (Figure 5B).
Confocal laser-scanning microscopy showed that the leaf cells of in vitro-cultured seedlings treated with individual AM-toxin-2 to AM-toxin-7 were different from those of the control. The mesophyll cells of control plants had a typical structure, with intact chloroplasts with green–red autofluorescence (Figure 5B). However, following treatment with each AM-toxin, the susceptible cultivar Gala-3 showed an abnormal mesophyll cell structure. Structural changes occurred in the plasma membrane, chloroplasts were damaged and lost their autofluorescence, and nuclear membranes were disrupted. We successfully stained the nuclei of leaf cells with PI, as evidenced by their red fluorescence, which is indicative of cell death. Chlorophyll autofluorescence disappeared at 48 h post toxin treatment (hpt) (Figure 5B). At 48 h after treatment with each AM-toxin (48 hpt), necrosis occurred in all leaves of the susceptible cultivar Golden Delicious but not in the control (uninoculated Golden Delicious seedlings) (Figure 5C).
These results indicate that toxins from ALT2 to ALT7 (AM-toxin-2 to AM-toxin-7, respectively) induce leaf-spot symptoms in apple leaves and destroy chloroplasts and nuclear membranes, followed by cell death.

3.5. Effects of Inactivated AM-Toxins on Apple Leaves

To further investigate the symptoms of the toxins produced by ALT, we used potassium permanganate (KMnO4) to inactivate each AM-toxin. When we treated Gala-3 leaves with the KMnO4-inactivated versions of AM-toxin-2–7, the leaves showed very weak responses. Confocal laser-scanning microscopy showed that at 48 hpt, mesophyll leaf cells of in vitro-cultured seedlings treated with the KMnO4-inactivated versions of AM-toxin-2–7 had a typical structure, with intact chloroplasts emitting autofluorescence similar to that of control Gala-3 plants (Figure 6A). Similarly, we observed no differences between control and treated leaves of the susceptible cultivar Golden Delicious. In treated Gala-3 and Golden Delicious leaves, we did not detect red fluorescence from the nucleus following PI staining, suggesting that the plasma and nuclear membranes were intact (Figure 6B). The chloroplasts from Gala-3 or Golden Delicious leaves inoculated with the KMnO4-inactivated versions of AM-toxin-2–7 were not damaged and remained intact (Figure 6).

3.6. Identification of ALT7 Secretory Proteins

ALT produces various effector molecules to modulate its infection, although not much is known about the components that are crucial for its pathogenicity. We performed LC–MS of ALT7 fluid secretion (Supplemental Figure S3A). We identified 13,466 peptide sequences that had a match in the ALT genome (GCF_001642055.1), with 243 protein sequences that exceeded the identification threshold. Using SignalP (https://services.healthtech.dtu.dk/services/SignalP-6.0/, 22 April 2023) to filter the 243 protein sequences obtained by MS, we identified 136 protein sequences containing a signal peptide. Furthermore, we used TMHMM (https://services.healthtech.dtu.dk/services/TMHMM-2.0/, 14 June 2023) to look for transmembrane domains: 134 proteins contained a signal peptide but lacked a transmembrane structure. These 134 proteins were judged to be secretory proteins.
The top 10 secretory proteins are listed in Table 3, and the LC–MS profiles of these fungal exosome proteins are shown in Supplemental Figure S3B. We selected three of these proteins for further analysis: the first protein on the list is the ABC-type Fe3+ transport system protein ABC (XP_018388203.1; the second protein is alcohol oxidase (AO; XP_018381613.1), and the fifth is phosphodiesterase/alkaline phosphatase D precursor (PDE; XP_018383919.1). To further study the effects of ALT7 secretory proteins, we over-expressed the genes encoding each ALT7 secretory protein in Hanfu leaves (lines OE-AltABC, OE-AltAO, and OE-AltPDE) and measured the expression of AltABC, AltAO, and AltPDE in the transgenic seedlings (Figure 7A). At 48 h after treatment, we observed significant changes in each plant (Figure 7B). The leaves appeared highly symptomatic, and necrosis appeared on all leaves of OE-AltABC, OE-AltAO, and OE-AltPDE plants but not control (WT and pFGC5941) plants (Figure 7B–D). When OE plants were inoculated with strain ALT7, they allowed the production of higher fungal biomass (Figure 7C) and increased lesion sizes (Figure 7D). Thus, these three proteins may contribute to fungal infection of ALT7 by interacting with the key components of the immune system in the host and impair their activities.

4. Discussion

Morphological identification can differentiate Alternaria leaf spot, Glomerella leaf spot, and Marssonina leaf spot to a certain extent, but further verification is needed using molecular identification methods [30,31]. In this study, we identified and analyzed apple leaf-spot disease in the Yantai apple-growing region in China. Among the three apple leaf-spot diseases, Alternaria leaf spot had the highest incidence in the Yantai area, followed by Marssonina leaf spot and Glomerella leaf spot. Glomerella leaf spot mainly infects Gala apple, and the samples collected in this study were of the Fuji variety, which is highly resistant to Glomerella leaf spot [32,33]. Therefore, in Fuji apple, Glomerella leaf spot had the lowest incidence in the Yantai area.
Alternaria spp. release host-specific toxins to aid its pathogenicity [34]. These toxins target the host cell and its organelles, including the Golgi complex, mitochondria, plasma membrane, nucleus, and metabolic pathways such as ceramide and ATP synthesis [34]. Most of the existing studies on the pathogenicity of apple leaf spot have focused on toxins, and several toxins, such as AM-toxin-1 and AM-toxin-4, have been isolated [35]. In addition to toxins, effector proteins are one of the major pathways of pathogenicity of pathogenic fungi. Currently, only effectors and cell wall lytic enzymes have been analyzed in Alternaria using bioinformatics, and there is no evidence for functional validation of these effector proteins [34]. However, there have been no studies on the secreted proteins of apple leaf spot and effector proteins, as one of the main pathways of infection of plants by the pathogen, have not yet been identified.
This is the first study to investigate the secretory proteins of ALT. First, we obtained six purified isolates of ALT from Fuji leaves in the apple production area. Following pathogenicity analysis, we extracted crude toxins from these strains and found that symptoms obtained with the extracted toxins (AM-toxin-2 to AM-toxin-7) were similar to those of the isolated fungal strains (ALT2 to ALT7), with symptoms appearing on each leaf. AM toxin is a specialized host toxin produced by A. alternata [22]. The fungal isolates were tested using specific primers encoding AM toxin (AMT1 and AMT4), and it was found that ALT7 released the highest amount of the toxin [22].In addition to producing leaf-spot symptoms, each AM toxin destroyed chloroplasts and nuclear membranes, whereas inactivated toxins lost most of their deleterious effects. Finally, since ALT7 showed the strongest pathogenicity, we performed LC–MS of ALT7 extracellular vesicles. We identified 134 proteins containing signal peptides and lacking transmembrane structures, which were judged to be secretory proteins. Among the top 10 secretory proteins, we selected the ABC-type Fe3+ transport system (XP_018388203.1), alcohol oxidase (XP_018381613.1), and phosphodiesterase/alkaline phosphatase D precursor (XP_018383919.1) for further study. We over-expressed the genes encoding these ALT7 secretory proteins in Gala-3 leaves, generating lines OE-Alt7ABC, OE-Alt7AO, and OE-Alt7PDE. The leaves appeared highly symptomatic, and necrosis symptoms appeared on all leaves of these plants but not control (WT and pFGC5941) plants. At 48 h after treatment, we observed significant changes in plants treated with each ALT7 secretory protein. These proteins may contribute to fungal infection by interacting with the key components of the immune system in the host and impairing their activities. Further studies on these phytotoxins and secretory proteins of ALT will provide a better understanding of apple–pathogen interactions.

5. Conclusions

We examined the incidence of leaf spot in Fuji apple trees in the apple-growing region in Yantai, Shandong Province, China. The incidence of Alternaria leaf spot was the highest, followed by Marssonina leaf spot and Glomerella leaf spot. We identified six Alternaria alternata f. sp. mali strains (ALT2 to ALT7) from 124 diseased Fuji leaf samples in Yantai. These strains exhibited pathogenicity to four apple cultivars with different levels of resistance, with ALT7 showing the strongest pathogenicity. ALT7 secretes elicitor proteins that act as virulence factors in its interactions with plants. We determined that the fungal-secreted proteins Alt7ABC, Alt7AO, and Alt7PDE contribute to fungal infection by ALT, and we will further explore the pathogenic mechanisms of three secreted proteins.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae10030212/s1, Figure S1: Map indicating the location of five districts in Yantai: Laishan District, Muping District, Haiyang City, Qixia City, and Zhaoyuan City; Figure S2: Growth status of hyphae and spores in apple leaves inoculated with Alternaria alternata var. mali strain ALT7 from 0 to 48 h post-inoculation (hpi). A. Staining of ALT7-inoculated Gala-3 leaves at 0 hpi, 24 hpi, and 48 hpi with aniline blue fluorochrome. B. Staining of ALT7-inoculated Golden Delicious leaves at 0 hpi, 24 hpi, and 48 hpi with aniline blue fluorochrome. C. Staining of ALT7-inoculated Shandong Gala leaves at 0 hpi, 24 hpi, and 48 hpi with aniline blue fluorochrome. D. Staining of ALT7-inoculated Hanfu leaves at 0 hpi, 24 hpi, and 48 hpi with aniline blue fluorochrome. Aniline blue fluorescence merged with DIC. Scale bars = 50 µm; Figure S3: Identification of secretory proteins from Alternaria alternata var. mali strain ALT7. A. Schematic of extraction of ALT2/3/4/5/6/7 exosomes. B. Top 10 secretory proteins. C. Analysis of the LC-MS data for top 10 secretory proteins; Table S1: Primers used in this study.

Author Contributions

Q.Z. and T.L. conceived and supervised the study. C.C. and S.G. performed the experiments. J.T. and Y.L. analyzed the data. Q.Z. wrote the article and did the final editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the 2115 Talent Development Program of China Agricultural University, the National Natural Science Foundation of China (Grant No. 32272640) and was supported by the Chinese Universities Scientific Fund (Grant No. 2023TC099).

Data Availability Statement

Data are contained within the article. Additional data can be obtained by contacting the corresponding author of the article.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Examples of apple leaf spot fungal strains in Yantai. The pathogenic fungi were isolated from diseased ‘Fuji’ leaves (top panels), inoculated onto potato dextrose agar medium, and observed from the top of each plate (middle panels); mycelial spore morphology was observed by microscopy after 6 days of culture (bottom panels).
Figure 1. Examples of apple leaf spot fungal strains in Yantai. The pathogenic fungi were isolated from diseased ‘Fuji’ leaves (top panels), inoculated onto potato dextrose agar medium, and observed from the top of each plate (middle panels); mycelial spore morphology was observed by microscopy after 6 days of culture (bottom panels).
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Figure 2. Morphological characterization and sequence analysis of single-spore isolates of Alternaria alternata f. sp. mali (ALT). (A). Strains ALT2/3/4/5/6/7 after 6 days of growth on potato dextrose agar. (B–D). Agarose gel electrophoresis of three marker loci of ALT2/3/4/5/6/7; M, BL2000 DNA marker; PC, positive control, ALT1 [8]; NC, negative control, ddH2O.
Figure 2. Morphological characterization and sequence analysis of single-spore isolates of Alternaria alternata f. sp. mali (ALT). (A). Strains ALT2/3/4/5/6/7 after 6 days of growth on potato dextrose agar. (B–D). Agarose gel electrophoresis of three marker loci of ALT2/3/4/5/6/7; M, BL2000 DNA marker; PC, positive control, ALT1 [8]; NC, negative control, ddH2O.
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Figure 3. Pathogenicity bioassays of Alternaria alternata f. sp. mali strains ALT2/3/4/5/6/7 on apple. (A). Phenotypes of 4-week-old Gala-3 (GL-3) at 2 days after inoculation with ALT2/3/4/5/6/7. (B). Lesion areas in Gala-3 leaves inoculated with ALT2/3/4/5/6/7 at 48 hpi. (C). Phenotypes of four-week-old GD (Golden Delicious) at 2 days after inoculation with ALT2/3/4/5/6/7. (D). Lesion areas in GD leaves inoculated with ALT2/3/4/5/6/7 at 48 hpi. (E). Phenotypes of 4-week-old SGL (Shandong Gala) at 2 days after inoculation with ALT2/3/4/5/6/7. (F). Lesion areas in SGL leaves inoculated with ALT2/3/4/5/6/7 at 48 hpi. (G). Phenotypes of 4-week-old HF (Hanfu) at 2 days after inoculation with ALT2/3/4/5/6/7. (H). Lesion areas in HF leaves inoculated with ALT2/3/4/5/6/7 at 48 hpi. Error bars = SD, three biological replicates. ** indicates a significant difference.
Figure 3. Pathogenicity bioassays of Alternaria alternata f. sp. mali strains ALT2/3/4/5/6/7 on apple. (A). Phenotypes of 4-week-old Gala-3 (GL-3) at 2 days after inoculation with ALT2/3/4/5/6/7. (B). Lesion areas in Gala-3 leaves inoculated with ALT2/3/4/5/6/7 at 48 hpi. (C). Phenotypes of four-week-old GD (Golden Delicious) at 2 days after inoculation with ALT2/3/4/5/6/7. (D). Lesion areas in GD leaves inoculated with ALT2/3/4/5/6/7 at 48 hpi. (E). Phenotypes of 4-week-old SGL (Shandong Gala) at 2 days after inoculation with ALT2/3/4/5/6/7. (F). Lesion areas in SGL leaves inoculated with ALT2/3/4/5/6/7 at 48 hpi. (G). Phenotypes of 4-week-old HF (Hanfu) at 2 days after inoculation with ALT2/3/4/5/6/7. (H). Lesion areas in HF leaves inoculated with ALT2/3/4/5/6/7 at 48 hpi. Error bars = SD, three biological replicates. ** indicates a significant difference.
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Figure 4. Relative expression of the toxin genes AMT1 and AMT4 in inoculated apple seedling leaves. (A). Relative expression of AMT1 and AMT4 in Alternaria alternata f. sp. mali strains ALT2/3/4/5/6/7-inoculated GL-3 (Gala-3). (B). Relative expression of AMT1 and AMT4 in ALT2/3/4/5/6/7-inoculated GD (Golden Delicious). (C). Relative expression of AMT1 and AMT4 in ALT2/3/4/5/6/7-inoculated SGL (Shandong Gala). (D). Relative expression of AMT1 and AMT4 in ALT2/3/4/5/6/7-inoculated HF (Hanfu). Error bars = SD, three biological replicates. ** indicates a significant difference.
Figure 4. Relative expression of the toxin genes AMT1 and AMT4 in inoculated apple seedling leaves. (A). Relative expression of AMT1 and AMT4 in Alternaria alternata f. sp. mali strains ALT2/3/4/5/6/7-inoculated GL-3 (Gala-3). (B). Relative expression of AMT1 and AMT4 in ALT2/3/4/5/6/7-inoculated GD (Golden Delicious). (C). Relative expression of AMT1 and AMT4 in ALT2/3/4/5/6/7-inoculated SGL (Shandong Gala). (D). Relative expression of AMT1 and AMT4 in ALT2/3/4/5/6/7-inoculated HF (Hanfu). Error bars = SD, three biological replicates. ** indicates a significant difference.
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Figure 5. Phenotypes and cellular effects of AM-toxins on apple leaves. (A). Schematic diagram of toxin extraction from Alternaria alternata f. sp. mali strains ALT2/3/4/5/6/7. (B). AM-toxin-2/3/4/5/6/7-damaged chloroplasts and plasma membranes in GL-3 (Gala-3) leaves. (C). AM-toxin-2/3/4/5/6/7-damaged chloroplasts and plasma membranes in GD (Golden Delicious) leaves. Propidium iodide was used to stain the leaves of control plants and leaves treated with AM-toxin-2/3/4/5/6/7. Confocal microscopy images show red fluorescence at 580–610 nm (representing propidium iodide-stained nuclei) and green fluorescence at 629–670 nm (representing autofluorescence of chloroplasts).
Figure 5. Phenotypes and cellular effects of AM-toxins on apple leaves. (A). Schematic diagram of toxin extraction from Alternaria alternata f. sp. mali strains ALT2/3/4/5/6/7. (B). AM-toxin-2/3/4/5/6/7-damaged chloroplasts and plasma membranes in GL-3 (Gala-3) leaves. (C). AM-toxin-2/3/4/5/6/7-damaged chloroplasts and plasma membranes in GD (Golden Delicious) leaves. Propidium iodide was used to stain the leaves of control plants and leaves treated with AM-toxin-2/3/4/5/6/7. Confocal microscopy images show red fluorescence at 580–610 nm (representing propidium iodide-stained nuclei) and green fluorescence at 629–670 nm (representing autofluorescence of chloroplasts).
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Figure 6. Cellular effects of inactivated AM-toxins from Alternaria alternata f. sp. mali strains ALT2/3/4/5/6/7 on apple leaves. (A). AM-toxin-2/3/4/5/6/7 did not damage chloroplasts or plasma membranes in GL-3 (Gala-3) after being inactivated by KMnO4. (B). AM-toxin-2/3/4/5/6/7 did not damage chloroplasts or plasma membranes in GD (Golden Delicious) after being inactivated by KMnO4. Confocal microscopy images show red fluorescence at 580–610 nm (representing propidium iodide-stained nuclei) and green fluorescence at 629–670 nm (representing autofluorescence of chloroplasts).
Figure 6. Cellular effects of inactivated AM-toxins from Alternaria alternata f. sp. mali strains ALT2/3/4/5/6/7 on apple leaves. (A). AM-toxin-2/3/4/5/6/7 did not damage chloroplasts or plasma membranes in GL-3 (Gala-3) after being inactivated by KMnO4. (B). AM-toxin-2/3/4/5/6/7 did not damage chloroplasts or plasma membranes in GD (Golden Delicious) after being inactivated by KMnO4. Confocal microscopy images show red fluorescence at 580–610 nm (representing propidium iodide-stained nuclei) and green fluorescence at 629–670 nm (representing autofluorescence of chloroplasts).
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Figure 7. Phenotypes and symptoms of three secretory proteins of Alternaria alternata f. sp. mali overexpressed (OE) in leaves of resistant Hanfu (HF). (A). Relative expression of AltABC, AltAO, and AltPDE in OE-AltABC-, OE-AltAO-, and OE-AltPDE. (B). Phenotypes of OE-AltABC-, OE-AltAO-, and OE-AltPDE HF. (C). Biomass strain of ALT7 in OE-AltABC-, OE-AltAO-, and OE-AltPDE seedlings after inoculation with ALT7. (D). Lesion area of OE-AltABC-, OE-AltAO-, and OE-AltPDE HF after inoculation with ALT7. Error bars = SD, three biological replicates. Different letters indicate significant differences.
Figure 7. Phenotypes and symptoms of three secretory proteins of Alternaria alternata f. sp. mali overexpressed (OE) in leaves of resistant Hanfu (HF). (A). Relative expression of AltABC, AltAO, and AltPDE in OE-AltABC-, OE-AltAO-, and OE-AltPDE. (B). Phenotypes of OE-AltABC-, OE-AltAO-, and OE-AltPDE HF. (C). Biomass strain of ALT7 in OE-AltABC-, OE-AltAO-, and OE-AltPDE seedlings after inoculation with ALT7. (D). Lesion area of OE-AltABC-, OE-AltAO-, and OE-AltPDE HF after inoculation with ALT7. Error bars = SD, three biological replicates. Different letters indicate significant differences.
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Table 1. Strain collection and analysis of apple leaf spot in Yantai.
Table 1. Strain collection and analysis of apple leaf spot in Yantai.
DistrictNumber of StrainsAlternaria Leaf Spot (Alternaria alternata f. sp. mali)Glomerella Leaf Spot (Glomerella cingulate)Marssonina Leaf Spot (Marssonina coronaria)
Laishan2318 (78.26%)06 (26.08%)
Muping2416 (66.67%)2 (8.33%)4 (16.67%)
Haiyang2316 (48.48%)07 (21.21%)
Qixia2015 (75%)1 (5%)14 (58.33%)
Zhaoyuan2419 (79.16%)5 (20.83%)13 (54.16%)
Table 2. GenBank accession numbers of strains ALT2/3/4/5/6/7. ITS: internal transcribed spacer; Beta-tubulin: tubulin gene; Alt a 1: Alt a 1 gene.
Table 2. GenBank accession numbers of strains ALT2/3/4/5/6/7. ITS: internal transcribed spacer; Beta-tubulin: tubulin gene; Alt a 1: Alt a 1 gene.
SpeciesStrain NumberITSBeta-TubulinAlt a 1
GenBank Accession NumbersPercent IdentityGenBank Accession NumbersPercent IdentityGenBank Accession NumbersPercent Identity
A.alternataALT2MF422130.198.95%KY814630.199.42%OP311599.199.54%
A.alternataALT3ON973886.199.63%ON667992.199.42%OR464084.198.44%
A.alternataALT4MZ930190.199.81%ON667994.199.42%OQ686979.199.54%
A.alternataALT5ON712167.199.82%KY814629.1100%OQ831518.1100%
A.alternataALT6KJ739872.199.12%ON667994.198.83%JQ282256.198.63%
A.alternataALT7MH553296.199.47%KY814628.199.22%OR061063.198.13%
Table 3. GenBank accession numbers of secretory proteins from exosomes of Alternaria alternata f. sp. mali strain ALT7.
Table 3. GenBank accession numbers of secretory proteins from exosomes of Alternaria alternata f. sp. mali strain ALT7.
FamilyAccessionScoreMassemPAIDescription
2XP_018388203.1136939,2941.91ABC-type Fe3+ transport system
3XP_018381613.1127369,0312.01alcohol oxidase
4XP_018391646.1111483,9110.74hypothetical protein CC77DRAFT_1015550
5XP_018384467.1977102,1060.51hypothetical protein CC77DRAFT_966271
7XP_018383919.192070,7170.27phosphodiesterase/alkaline phosphatase D precursor
8XP_018382257.186149,5280.41hypothetical protein CC77DRAFT_1011906
10XP_018384413.173833,6890.86alpha/beta-hydrolase
11XP_018382523.164544,5640.76glycoside hydrolase
13XP_018387936.159240,6640.68hypothetical protein CC77DRAFT_733950
14XP_018390870.157156,3460.96FAD-binding domain-containing protein
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Cao, C.; Gong, S.; Li, Y.; Tang, J.; Li, T.; Zhang, Q. Pathogenic Factors and Mechanisms of the Alternaria Leaf Spot Pathogen in Apple. Horticulturae 2024, 10, 212. https://doi.org/10.3390/horticulturae10030212

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Cao C, Gong S, Li Y, Tang J, Li T, Zhang Q. Pathogenic Factors and Mechanisms of the Alternaria Leaf Spot Pathogen in Apple. Horticulturae. 2024; 10(3):212. https://doi.org/10.3390/horticulturae10030212

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Cao, Chen, Shun Gong, Yan Li, Jinqi Tang, Tianzhong Li, and Qiulei Zhang. 2024. "Pathogenic Factors and Mechanisms of the Alternaria Leaf Spot Pathogen in Apple" Horticulturae 10, no. 3: 212. https://doi.org/10.3390/horticulturae10030212

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Cao, C., Gong, S., Li, Y., Tang, J., Li, T., & Zhang, Q. (2024). Pathogenic Factors and Mechanisms of the Alternaria Leaf Spot Pathogen in Apple. Horticulturae, 10(3), 212. https://doi.org/10.3390/horticulturae10030212

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