Insights into the Adaptation of Geotrichum citri-aurrantii in Highly Acidic Environments

TL;DR

This study explores how the fungus Geotrichum citri-aurantii adapts to acidic citrus environments, revealing key genetic and structural changes that help it survive and cause sour rot.

Contribution

The study identifies novel adaptive mechanisms and gene regulation patterns in G. citri-aurantii under acidic conditions, offering new insights into its pathogenicity.

Findings

G. citri-aurantii modifies host pH through alkalizing or acidifying depending on initial acidity.

Transcriptomic analysis reveals regulation of genes involved in cell wall remodeling and carbon metabolism under acid stress.

The Pal/Rim pH signaling pathway shows distinct responses in citrus cultivars with varying acidity levels.

Abstract

Sour rot is a significant postharvest disease affecting citrus fruit, causing sourness and decay in various cultivars, particularly lemons. How the pathogen, Geotrichum citri-aurantii, adapts to the highly acidic environment of citrus fruit remains inadequately understood. In this study, the growth characteristics, morphological and structural changes, gene expression profiles, and adaptive mechanisms of G. citri-aurantii under highly acidic conditions were elucidated. The findings indicated that G. citri-aurantii modified the environmental pH by either alkalizing (pH < 3.00) or acidifying (pH > 3.00) the host tissue. It exhibited strong adaptability at pH 2.2, showing shortened and aggregated hyphae, delayed spore germination, and increased vacuoles. Transcriptomic analysis and qRT-PCR identified the significant regulation of key differentially expressed genes involved in cell wall remodeling, cell membrane component synthesis, carbon metabolism, and signal transduction. These regulatory changes enable the pathogen to prevent an influx of external acids and maintain the energy supply under acid stress conditions. Additionally, the Pal/Rim pH signaling pathway genes exhibit distinct response patterns in citrus cultivars with different acidities. These findings enrich the comprehension of the pathogenic process of G. citri-aurantii and offer a theoretical foundation for preventing and managing citrus sour rot.

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1. Introduction

In southern China, citrus is the most extensively grown tree fruit crop [1]. Citrus fruits are mainly consumed fresh, and they can rot and deteriorate due to fungal infections during storage and transportation, causing economic losses and adversely affecting citrus industry development [2]. The primary citrus pathogens—Penicillium italicum (blue mold), P. digitatum (green mold), and Geotrichum citri-aurantii (sour rot)—are notorious for causing postharvest diseases in stored and transported fruit [3]. Among them, citrus sour rot is likely to occur during heavy rain. The rotten part of the diseased fruit features a layer of white mold and emits a sour and foul smell. Despite being approved for citrus postharvest decay management, fungicides such as thiabendazole and imazalil are not effective against G. citri-aurantii. The options for prevention and control are quite limited [4]. Therefore, exploring the pathogenic characteristics of G. citri-aurantii is particularly important for understanding its impact and developing effective control strategies.

The living activities of pathogenic fungi are closely related to environmental pH, which is a crucial environmental factor affecting pathogenic factors like cell wall-degrading enzymes and mycotoxins [5,6]. In our previous report, G. citri-aurantii was found to grow best at a pH of around 3.0, and it can infect citrus fruit with varying acidity levels. In tangerine varieties, G. citri-aurantii can reduce the pH value at the infection site [7]. Similarly, P. digitatum, P. italicum, Sclerotinia sclerotiorum, and Botrytis cinerea have been found to down-regulate the pH value of host tissues by secreting organic acids, which are identified as acidic fungi [6]. However, there are a few pathogenic fungi that can thrive on lemon fruit with a pH of only 2.2 as effectively as G. citri-aurantii [7].

Citrus fruits contain a high concentration of organic acids, with citric acid making up 60–90% of the total and being the main acid present [8]. Increased acidity in the microbial growth environment typically results in higher intracellular acidity and accelerates cells’ metabolic disorders [5]. Under extremely acidic conditions, the intracellular pH of many filamentous fungi remains nearly neutral, due to factors such as cell membrane stability, intracellular buffering, and DNA and protein repair [9]. The tolerance of Saccharomyces cerevisiae to lactic acid involves gene expression changes in areas such as amino acid and energy metabolism, anion transport, maintaining intracellular pH, and rearranging the cell envelope [10]. In P. expansum, gene expression regulation in response to ambient pH is significantly influenced by the Pal/Rim-pH pathway. When the dominant transcription factor of this pathway, PacC, is knocked out, P. expansum exhibits limited growth in both acidic and alkaline environments [11]. At present, the adaptability of G. citri-aurantii to different environmental pH values has been confirmed to be related to the expression of the transcription factor pacC [7]. However, the regulatory mechanism of acid adaptability has not yet been revealed.

This study investigates the environmental acidification capacity and acid stress adaptability of G. citri-aurantii through in vitro and in vivo experiments. The most suitable pH for growth and the most extreme acidic pH that can be accommodated were identified for fungal cell culture. Subsequently, the adaptation mechanism of G. citri-aurantii under highly acidic conditions was investigated using electron microscopy and transcriptomic analysis. The results could enrich research on the pathogenic mechanism of G. citri-aurantii and provide a theoretical basis for managing citrus sour rot.

2. Materials and Methods

2.1. Lesion Diameter and pH Change in Decaying Areas of Different Citrus Varieties

Eureka lemon, Sour orange, and Satsuma mandarin of the same maturity and size were selected as materials. The citrus fruits were washed and sterilized in a 2% NaClO solution. To amplify the pH variation in the diseased area, two cross-shaped wounds (3 mm deep and 8 mm wide) were created in the equatorial region of the fruit using a sterilized scalpel. This was followed by inoculating 10 μL of a spore suspension at a concentration of 1 × 10^7^ spores/mL, which was injected into the center of each wound. A water treatment was used as a control. An incubator maintained at 28 ± 2 °C and 85–90% relative humidity was used to store the fruit [12]. The lesion diameter and pH change in the decaying area were measured with an interval of 1 d.

2.2. Change in pH Value Under Different Acidic Conditions

To simulate the potential pH values found in various citrus varieties, the PDB culture medium’s pH was modified with citric acid to reach initial pH values of 2.01, 3.02, 4.03, and 4.99, respectively. The pH level of the culture medium was recorded after 2 days of cultivation with 1 mL of the spore suspension [7].

2.3. G. citri-aurantii Growth in Different Media and Influence on pH Value

Following a 4-day incubation on PDA plates, the spores were rinsed off with sterile water, filtered through four layers of gauze, and adjusted to 5 × 10^7^ spores per milliliter conidial suspension. A spore suspension of 1 mL and 0.1 mL was mixed with 49 mL and 49.9 mL of IM medium, respectively, which contained 1% K-buffer (pH 4.8–4.9), 0.1% CaCl_2_, 0.2% glucose, 0.06% MgSO_4_·7H_2_O, 0.03% NaCl, 0.25% (NH_4_)2_SO_4, and 0.5% glycerol [13]. The resulting solution was then set to a temperature of 28 ± 2 °C with continuous shaking. The pH value of the medium was tested at each interval, and the precipitate was collected by centrifugation to determine the dry weight of the mycelium.

2.4. G. citri-aurantii Growth Under Different Acidic Conditions

The pH values of the PDB medium were adjusted to 1.0, 2.0, 2.2, 2.5, 3.0, 4.0, and 5.0 using citric acid or HCl (control). The spore suspension was added and maintained at 28 ± 2 °C with agitation. The spore germination rate was recorded by observation under a microscope at 3, 6, and 9 h, respectively. Additionally, the mycelia’s dry weight was assessed at 2 d [7].

2.5. Observing G. citri-aurantii Morphology and Structure Under Different pH Conditions

Given the strong viability of G. citri-aurantii in lemon fruit, we selected a pH value of 2.2 for the experimental group and examined the cytological and transcriptomic changes in the fungus compared to those at the optimal growth pH value of 3.0.

The PDB medium with pH values of 2.2 and 3.0 received a spore suspension with a final concentration of 1 × 10^6^ spores/mL (using citric acid to adjust the pH), and the mixture was incubated for 6 h (28 ± 2 °C, 160 rpm). Similarly, the morphology and structure of the mycelium were observed after 1 d of culture. Microscopic observation was conducted on aliquots of the suspension placed on glass slides. After being treated with an ethanol concentration gradient, freeze-drying, and gold coating, these samples were prepared for SEM observation (JSM6610LV, JEOL Ltd., Tokyo, Japan) [14]. The intracellular changes in the cells were examined using a transmission electron microscope (JEM-1200EX; JEOL Ltd., Tokyo, Japan) with an accelerating voltage of 80 kV [12].

2.6. Transcriptomic Analysis of G. citri-aurantii Under Acidic Conditions of pH 2.2 and 3.0

A final concentration of 10^6^ spores/mL was added to the PDB medium with the above pH values and cultured at 28 ± 2 °C for 24 h with a rotational speed of 160 r/min. Samples were washed thrice with PBS (0.1 mol/L, pH 6.8) and centrifuged (4000 rpm, 4 °C, 10 min). The precipitates were gathered and sequenced through Illumina HiSeq2500 (Gene Denovo Biotechnology Co., Guangzhou, China). Functional genes and pathways were identified via GO and KEGG databases, and Cytoscape version 3.7.1 software was employed to analyze the networks of differentially expressed genes (DEGs) [15]. An analysis of the PPI network of the DEGs was performed with interaction data from the STRING database (http://string-db.org/, accessed on 8 December 2022).

2.7. qRT-PCR Verification for Key DEGs

RNA extraction and reverse transcription were conducted on samples taken at 0, 6, 12, and 24 h. SYBR Green real-time PCR was carried out with an ABI 2720 thermal cycler (Applied Biosystems, Waltham, MA, USA), and the primer sequences are detailed in Supplementary Table S1. The relative gene expression levels were calculated using the 2^−ΔΔCT^ method [15].

2.8. Statistical Analyses

The results presented include the mean ± standard error of the mean. Statistical analyses and figure generation were conducted using SPSS 19.0 (IBM Corp., Armonk, NY, USA), Origin 8.5 (OriginLab, Northampton, MA, USA), and Microsoft Excel 2013. To determine significant differences in values, Duncan’s multiple range test (p < 0.05) was conducted via a one-way ANOVA.

3. Results

3.1. Disease Development and pH Change in Different Citrus Varieties

Under the same inoculation conditions, the Satsuma mandarin fruit artificially exhibited the fastest disease onset. The water-stained lesion appeared on the fruit at 1 day post-inoculation (dpi), with a diameter of 0.7 ± 0.05 cm, while an obvious lesion appeared on the Eureka lemon and Sour orange at 2 dpi. From 2 to 7 dpi, the incidence rates of the three citrus varieties were similar, indicating that the initial fruit pH value only affects the early stage of G. citri-aurantii infection (Figure 1A).

Among the citrus varieties tested, Eureka lemon fruit had a pH value of 2.21 ± 0.03, Sour orange fruit had a pH of 2.59 ± 0.01, and Satsuma mandarin fruit had a higher pH value (3.29 ± 0.07). The pH value of the diseased part of the fruit with an initial pH < 3.0 increased, while the pH of the decaying area with a pH value > 3.0 decreased. Among them, the decaying area of the Eureka lemon and Sour orange fruit was markedly upregulated on inoculation days 2 and 3, respectively. However, in the Satsuma mandarin fruit, the pH value was significantly downregulated on inoculation day 4 (Figure 1B).

3.2. pH Value of Culture Under Different Acidic Conditions

Only the pH of the PDB broth with a pH of 2.0 was increased after 2 d of culture, while the pH of the PDB broth with a pH of 3.0 to 5.0 was decreased (Figure 2). The results showed that G. citri-aurantii modified the environmental pH by increasing it when the pH was below 3.02 and reducing it when the pH was above 3.02. For G. citri-aurantii, a pH level of 2.01 to 3.02 is more conducive to growth.

3.3. G. citri-aurantii Growth in Different Media and Influence on pH Value

The development of G. citri-aurantii in PDB and IM medium, along with its impact on the pH levels of the culture medium, exhibited varying patterns. G. citri-aurantii remained in the lag phase from day 0 to day 1 in the PDB, with an initial pH value of 5.83. The medium’s pH dropped to 3.68 during the 1–2 d culture period in the logarithmic growth phase. The medium’s pH increased to 5.48, and during the stable and declining phases, it remained around 5.90 (Figure 3A,B). In the IM medium with an initial pH value of 5.49, G. citri-aurantii was in the lag stage from 0 to 8 d. When cultured for 8–12 d in the logarithmic growth stage, the medium’s pH dropped to 2.39. A significant decrease in pH was observed in the medium with 1.0 mL spore suspension, which was faster than in the medium with 0.1 mL. The medium’s pH rose to 2.30, and during the stable and declining stages, it stabilized at about 2.23 (Figure 3C,D). These results indicate that G. citri-aurantii can acidify the environment and has a high tolerance for a pH 2.2 environment. The behavior of G. citri-aurantii in environments with a changing pH may be related to the culture substrate.

3.4. G. citri-aurantii Growth in Different Acidic pH Ranges

After HCl and citric acid were used to adjust the medium’s pH, it was found that G. citri-aurantii exhibited maximum mycelial growth at pH 3.0, followed by pH 4.0 and 5.0. Mycelia did not grow significantly at pH levels of 1.0 to 2.0 (Figure 4A). The spore germination rate in the pH 3.0 environment was the highest at 3 h, but the spore germination rate in the pH 3.0–5.0 environment was not significantly different at 6–9 h. In media with pH values less than 3.0, lower pH values corresponded with a decreased spore germination rate. It is worth noting that spores still exhibited about a 10% germination rate in a pH 2.0 environment (Figure 4B), indicating that spores have stronger acid resistance than mycelia. Moreover, the mycelial growth of G. citri-aurantii was more inhibited by citric acid than by HCl in media with a pH lower than 2.2, whereas mycelia grew better in media with citric acid compared to those with HCl when the pH was 2.2 or higher (Figure 4A). Likewise, the spore germination rate was higher in a citric acid medium than a HCl medium, which is especially evident at 3 h (Figure 4B).

3.5. Microscopic Observation of G. citri-aurantii Under Different pH Conditions

To explore the adaptive ability of G. citri-aurantii in a pH 2.2 environment, we compared the differences in mycelial morphology and structure with those in a pH 3.0 environment. Under an optical microscope, the mycelia of the pH 3.0 medium cultured for 1 d grew vigorously, with long and full morphology, while the pH 2.2 medium exhibited considerably fewer and shorter hyphae (Figure 5A,B).

After cultivation in media at pH = 3.0 for 6 h, most of the spores had germinated. When cultured for 24 h, the mycelium was smooth and full, and the ends of the hyphae broke off to form arthrospores. When cultured at pH 2.2 for 6 h, most spores did not germinate, and depressions were observed on the surfaces of some spores. When cultured for 24 h, the mycelia became rough, and most of them did not fracture (Figure 5C–F).

After 24 h of cultivation at pH 3.0, G. citri-aurantii’s internal organelle structure was well-maintained, and its cell wall and membrane were intact and clearly outlined. Although the internal organelles of the mycelium cultured at pH 2.2 were intact, the enlarged vacuoles and the diffused edge of the cell wall were observed (Figure 5G,H).

3.6. Transcriptomic Analysis

Principal component analysis (PCA) showed that the transcriptome from the pH 2.2 treatment clearly segregated from that of the pH 3.0 treatment (Figure 6A). Culturing mycelia at pH 2.2 for 24 h resulted in 1626 differentially expressed genes, with 1021 genes being upregulated and 605 down-regulated (Figure 6B). GO and KEGG pathway enrichment analyses were conducted to better comprehend the functions of the DEGs. Several GO categories were enriched within the dataset, including terms such as ribosome structure (ribosomal subunit, GO:0044391; cytosolic ribosome, GO:0022626; ribosome, GO:0005840; ontology: cellular component), cell wall (fungal-type cell wall, GO:0009277; cell wall, GO:0005618; ontology: cellular component), and protein localization and targeted trafficking (establishment of protein localization to membrane, GO:0090150; protein localization to endoplasmic reticulum, GO:0070972; cotranslational protein targeting to membrane, GO:0006613; ontology: biological process). DEGs were also enriched in molecular function categories such as structural constituent of ribosome (GO:0003735) (Figure 6C).

KEGG annotations were allocated to 20 different classes, predominantly involving metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems. In particular, for metabolism, DEGs were associated with the metabolism of carbohydrates, amino acids, lipids, and energy. Other KEGG categories suggested that the functional regulation of DEGs might be linked to translation, folding, sorting and degradation, transport and catabolism, and signal transduction (Figure 6D). Overall, these findings emphasize the diverse and intricate functional adaptations of G. citri-aurantii to acid stress.

3.7. PPI Network Construction

All differentially expressed genes were analyzed for their protein network interactions. The darker the color, the greater the connectivity, and the more significant its role in this interaction network. As can be seen from the six key sub-networks, the most important proteins include the amino acid permease transporter (GAP1; degree, 40), ammonium permease involved in regulating pseudohyphal growth (MEP2; degree, 30), fructose-1, 6-diphosphate aldolase (FBA1; degree, 14), and cell-specific secreted protein (DSE4; degree, 10) (Figure 7). The expression levels of these genes were further verified at different incubation times. Additionally, these sub-network genes revealed that under a pH of 2.2, substantial changes occurred in the regulatory pathways of G. citri-aurantii, such as nitrogen metabolism, carbon metabolism, cell wall synthesis and remodeling, and nucleotide and amino acid biosynthesis.

3.8. qRT-PCR Validation

By combining KEGG, GO, PPI, and differential expression analyses, 20 key DEGs were identified. Quantitative PCR was then performed. Among the genes related to the cell wall, the expression levels of both DSE4, which encodes a protein similar to glucanases, and CTS1, which encodes endochitinase, decreased at 12–24 h (Figure 8A,B). ECM33, which encodes a GPI-anchored protein, dropped at 6–24 h (Figure 8C). At 12 h, the expression of GAS5, responsible for encoding 1,3-beta-glucanosyltransferase, was reduced, but it increased at 24 h (Figure 8D), while the expression of TOS1, encoding a covalently bound cell wall protein, increased at 6–24 h (Figure 8E). Among the genes related to the cell membrane, ATG22, responsible for encoding a vacuolar integral membrane protein necessary to release amino acids during the breakdown of autophagic bodies in the vacuole, was notably upregulated between 6 and 24 h, showing an increase of nearly tenfold (Figure 8F). The expression of genes associated with carbon metabolism and energy metabolism, including FBA1 (fructose 1,6-bisphosphate aldolase), SFC1 (Mitochondrial succinate-fumarate transporter), ALD4 (mitochondrial aldehyde dehydrogenase), and SDH2 (iron–sulfur protein subunit of succinate dehydrogenase), was downregulated initially and then upregulated (Figure 8G–J). Both ALDH3A1 (putative fatty aldehyde dehydrogenase) and FDH1, encoding NAD(+)-dependent formate dehydrogenase, were highly expressed at 24 h (Figure 8K,L). Only PDC1 (the major of the three pyruvate decarboxylase isozymes) was downregulated at 6–24 h (Figure 8M). For genes related to nitrogen metabolism, the expression levels of MEP2 (ammonium permease involved in regulation of pseudohyphal growth) and UGA4 (a GABA transport protein involved in utilizing GABA as a nitrogen source) were downregulated at 12 h and 6 h, respectively, and then both were upregulated by 2.5 to 3.0 times at 24 h. While DIP5 (dicarboxylic amino acid permease) was upregulated at both 6 and 24 h, the expression of gatA (4-aminobutyrate aminotransferase) also showed a slight upregulation at 6 to 24 h (Figure 8N–Q). Moreover, we also observed the downregulation of the stress-related gene GRE2 (NADPH-dependent methylglyoxal reductase) at 12–24 h, as well as the upregulation of signal transduction-related genes GAP1 (putative Ras GTPase-activating) and IRA2 (GTPase-activating protein that negatively regulates RAS by converting it from the GTP- to the GDP-bound inactive form) at 24 h (Figure 8R–T).

3.9. Expression Analysis of Genes Related to pH Signaling Pathways

As can be seen from Figure 9A, under in vitro culture conditions, the expression levels of PalA, PalF, PalH, PalI1, PalI2, and PacC1 were downregulated, while the expression of PalB and PalC was slightly upregulated. Notably, the expression level of PacC2 increased by 3.58-fold. Taking the expression at 2 d as a control, during the disease process of the Satsuma mandarin fruit, PalA was only upregulated at 4 d, while the expression levels of PalB, PalH, and PacC2 were only upregulated at 6 d. PalC, PalF, PalI1, and PacC1 were continuously upregulated from days 4 to 6. Notably, PalI2 expression levels rose by over sixfold (Figure 9B). During the disease process in the lemon fruit, PalA, PalF, PalH, PalI1, and PalI2 were all down-regulated, while only PacC1 was upregulated at 4 d. At 6 d, the expression levels of PalB, PalC, PalH, PalI2, PacC1, and PacC2 increased by varying degrees. Among them, PalB and PalH showed the most significant upregulation, increasing by 3.69-fold and 5.35-fold, respectively (Figure 9C).

4. Discussion

The morphological changes, spore germination, and host penetration in pathogenic fungi can be influenced by ambient pH levels through the regulation of enzyme activities, alteration of carbohydrate metabolism, and disruption of protein synthesis [16]. In a report by Wang et al. [16], pear (pH 4.98), apple (pH 3.72), and kiwifruit (pH 3.31) were used for inoculation with P. expansum. The results revealed that a low pH could restrict P. expansum growth and prevent patulin accumulation in the diseased portions. Our study found that ambient pH affected the growth rate of G. citri-aurantii (Figure 4), whereas fruit tissue pH only influenced its initial stage of infection (Figure 1). This indicates that G. citri-aurantii can avoid the impact of ambient pH by modulating it to become closer to the optimal pH for growth. A similar phenomenon was also found in other postharvest pathogens such as B. cinerea, Colletotrichum gloeosporioides, and Sclerotinia sclerotiorum [17,18,19]. Interestingly, the pH changes induced by G. citri-aurantii were distinctly different in various culture media. Bi et al. [20] noted that external carbon affected fungal metabolism and thereby regulated the dynamic pH changes in their microenvironment. High glucose in PDB medium boosted mycelium growth and acid production, resulting in a rapid decrease in the pH value of the medium, while reduced glucose led to ammonia accumulation and pH rebound [21]. However, under nutrient-limiting conditions, fungi recycle substances via autophagy, maintaining acid production [22]. This may explain the differences in the pH regulation of G. citri-aurantii in the two media. Thus, we speculated that pH regulation by G. citri-aurantii appears to be related to culture substrates.

As is well known, citric acid is the main acid in citrus fruits, and is essential in the tricarboxylic acid (TCA) cycle for fungal growth and metabolism [23,24,25,26]. Some fungi can utilize the energy and carbon from citrate decomposition to synthesize necessary substances [27]. This may account for the stronger adaptability of G. citri-aurantii to the citric acid environment than to other acidic conditions at the same pH value, as illustrated in Figure 4. However, when the citric acid concentration is relatively high, the growth of G. citri-aurantii was still impaired (Figure 5). Under suboptimal growth conditions, some fungi aggregate to form a “nutrient pool” to obtain nutrients through the recycling of nitrogen-containing minerals [28]. In acidic environments, Aspergillus sp. releases more polysaccharide secondary metabolites, which function as “adhesives” to enhance mycelium aggregation [29]. Indeed, we found that genes linked to glycoprotein assembly, as well as secretion and transport, were upregulated (Supplementary Table S2), explaining the mycelial aggregation. Moreover, the cell wall and cell membrane of fungi are highly adaptable structures that react to stress by changing their composition [30,31]. For example, chitin accumulation in the cell wall is a frequent response of various fungi to cellular stress [32]. Increased sterols, sphingolipids, and unsaturated fatty acids lower membrane fluidity and permeability, limiting acid diffusion [33,34,35]. Based on the results, the upregulation of GAS5, KRE9, UTR2, and TOS1 (Supplementary Table S2, Figure 8), and the downregulation of DSE4, CTS1, and GAS5 implied that G. citri-aurantii not only enhances cell wall β-glucan assembly but also blocks chitin transfer to β-glucans. This helps cells resist damage by repairing broken glucan networks [36,37]. Meanwhile, the upregulated expression of ergosterol synthesis-related genes (ERG2, ERG6, ERG7, ERG24) and the sphingolipid formation-related gene ELO2 suggests that G. citri-aurantii enhances the biosynthesis of sterols and sphingolipids. This is consistent with the membrane component changes that occur in response to the acid tolerance of S. cerevisiae [38]. Combined with the downregulated expression of PMA1 (the gene encoding plasma membrane V-ATPases that mediate proton efflux from the cytosol) (Supplementary Table S2), we propose that the alterations in the cell wall and cell membrane of G. citri-aurantii largely prevented cytoplasm acidification.

Furthermore, in response to acid stress, fungal vacuoles undergo division and proliferation, which helps distribute them across the cell and supports important functions like cell signaling, repairing the plasma membrane, and communication between organelles. The H^+^ electrochemical gradient, sustained by the vacuolar-type H^+^-ATPase, is integral to this division process [39,40]. In this study, an upregulation of PKR1 (the gene encoding the V-ATPase assembly factor) (Supplementary Table S2) and signal transduction-related genes GAP1 and IRA2 (Figure 8), along with an increase in vacuole numbers within fungal cells (Figure 5G), supported enhanced vacuolar fission and inter-organelle communication. Actually, the Pal/Rim pH signaling pathway has been identified to be associated with the fungal adaptation to environmental pH under alkaline and neutral conditions [6,41,42]. This pathway includes seven proteins: PalA, PalB, PalC, PalF, PalH, PalI, and the core transcription factor PacC [43,44]. In this experiment, both in vivo and in vitro results demonstrated that the Pal/Rim pathway is also involved in the response of G. citri-aurantii to acidic environments (Figure 9). He et al. [44] noted that the expression patterns of PalA/B/C vary among fungal species. However, our study found that even within the same species, the expression levels of PalA and PalC varied under different acidic conditions; the same pattern was also observed for PalF, PalH, PalI1, and PalI2. Interestingly, the two PacC genes responded consistently in different citrus cultivars, with PacC1 upregulated earlier (Figure 9), possibly due to differences in isoelectric points, phosphorylation sites, and structural features [7].

In highly acidic environments, fungal respiration is typically inhibited. However, fungi can adjust their carbon metabolism to maintain ATP production and sustain their energy supply [45,46]. For instance, under sorbic acid stress, S. cerevisiae reduces aerobic respiration, using glycerol as a substrate and relying on fermentation to supply energy [46]. In this study, we observed that FDH1 expression was upregulated (Figure 8), suggesting that G. citri-aurantii enhanced the crosstalk between one-carbon metabolism and energy metabolism. Furthermore, alterations in the expression profiles of genes associated with glycolysis and the TCA cycle imply an accumulation of pyruvate (Figure 8). This accumulation may facilitate the scavenging of stress-induced reactive oxygen species (ROS) in cells [47].

5. Conclusions

During years of substantial rainfall, citrus sour rot frequently results in significant fruit losses. None of the fungicides currently approved for use is capable of effectively managing this disease. Considering that citrus cultivars with varying acidity levels, including high-acidity varieties such as lemons, are all vulnerable to G. citri-aurantii, this study aims to elucidate the acidophilic and acid-tolerant characteristics of the pathogen. The pathogen fortifies the physical structure of its cell wall to prevent acid influx while simultaneously enhancing signal transduction and optimizing carbon metabolic pathways, as well as energy metabolism processes, thereby facilitating adaptation to acidic conditions. Additionally, the Pal/Rim pH signaling pathway may contribute to regulating environmental pH in response to the pathogen. These findings offer potential targets for the development of new fungicides.