First Report of the Anthracnose Pathogenic Agent on Walnut Fruits in China and Exploration of Its Biological Characteristics

Abstract

Anthracnose is recognized as a significant agricultural disease. This study investigates the disease symptoms characterized by black dots on walnut fruits observed in the walnut orchards of Longnan City, Gansu Province, China, in June 2022. These symptoms resemble those of anthracnose reported in previous studies. A strain designated Ht-10 was initially isolated and identified as belonging to the Colletotrichum species based on its morphological features. Pathogenicity tests confirmed that this strain induced pronounced anthracnose symptoms in walnuts, consistent with those originally observed in the field. Subsequently, multilocus phylogenetic analysis, which included partial sequences of the internal transcribed spacer (ITS), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin2 (TUB2), and chitin synthase (CHS-1) genes of Ht-10, indicated that it most likely clustered with Colletotrichum fioriniae. The determination of biological characteristics revealed that the optimal temperature for the growth of Ht-10 was 25 °C in full light at a pH of 6, with soluble starch and yeast paste serving as the optimal carbon and nitrogen sources, respectively. To our knowledge, this is the first report of C. fioriniae as a causal agent of anthracnose in walnut fruits in China.

1. Introduction

Walnut (Juglans regia L.), belonging to Juglandaceae [1], originates from ancient Persia [2] and is one of the oldest tree species known to humanity [3,4]. It is widely cultivated across temperate regions for its edible nuts and high-quality wood [5]. Walnuts, along with almonds, cashews, and hazelnuts, are recognized as one of four major tree nuts globally [6]. Known for their high content of fats, proteins, vitamins, and minerals [7], walnuts hold significant economic and healthcare value. They can be processed into walnut oil and various health care products [8,9]. As the largest global walnut producer, China accounted for half of the world’s production in 2020 [10]. As one of the primary regions of origin, walnuts have been extensively cultivated in China for thousands of years, and walnut production in the country has seen rapid development in recent years. Both the planting area and production of walnuts in China are the highest in the world [11,12]. Longnan City in Gansu Province is a key walnut production area, serving as the largest walnut planting base in the province. This region features extensive planting areas and high production, making it a vital economic resource for local residents.

Currently, walnut diseases characterized by walnut blight are primarily caused by Xanthomonas arboricola pv. juglandis [13]. Additionally, walnut anthracnose is attributed to several pathogens, including members of the Colletotrichum gloeosporioides species complex [14], Marssonina juglandis [15], Colletotrichum fructicola [16], and Colletotrichum siamense [17]. Walnut brown spot disease is caused by Fusarium spp., Alternaria spp., Phomopsis sp., Cladosporium sp., and Colletotrichum sp. [18]. Among these, anthracnose poses the most significant threat to walnut plants. As the area dedicated to walnut cultivation expands, the prevalence and diversity of these diseases have intensified, adversely affecting both the quality and yield of walnuts. Colletotrichum, a prominent genus of plant pathogenic fungi, is responsible for widespread diseases across various hosts, often resulting in symptoms such as leaf rot, fruit rot, and plant necrosis [19]. In Gansu Province, walnut production and its economic value experience substantial losses each year due to damage inflicted by diseases and pests, which are the primary factors hindering the advancement of the walnut industry.

Research within the domestic academic community regarding walnuts primarily focuses on several key aspects, including the collection and breeding of walnut germplasm resources [20], the prevention and control of pests and diseases [21,22], the functional analysis of compound components [23], and industrial development [24]. Over the past decade, walnut anthracnose has been reported in multiple countries, including the United States, France, Iran, and Turkey, causing long-standing yield and quality losses. Previous studies have primarily focused on the occurrence and prevention of walnut anthracnose, including prevention methods, control measures, and comprehensive strategies for management. In China, the existing reports regarding the pathogen of walnut anthracnose predominantly identify C. gloeosporioides, along with C. siamense and C. fructicola [25]. Chen Yaonian et al. [26] conducted investigations that revealed eight common diseases in walnut orchards in Longnan, four of which have a broad occurrence range and cause significant damage, categorizing them as major diseases. Among these significant diseases, three are fungal—namely, anthracnose, brown spot disease, and plaster disease—while one is bacterial, identified as walnut blight disease. Researchers have undertaken pathogen identification work for anthracnose in walnut-growing regions across Henan, Hubei, Shandong, Shaanxi, Hebei, and Beijing. However, there have been no detailed pathogen identification reports from other regions [16,27]. Despite pathogen characterization in Henan and Shanxi provinces, the dominant Colletotrichum species in Gansu’s unique microclimate remain unconfirmed, hindering region-specific control strategies.

Therefore, the objective of this study is to identify the species of the pathogen causing walnut fruit anthracnose in Gansu Province, China, based on its morphological and molecular biological characteristics. Furthermore, this research aims to conduct an in-depth investigation into the biological characteristics of the pathogen.

2. Materials and Methods

2.1. Fungal Isolation and Purification

In June 2022, symptomatic walnut fruit samples with black dots were collected from Longnan, Gansu, China (105°36′53.74″ E, 33°39′11.14″ N). Samples were washed with tap water, then cut into 2–3 mm thick pieces. They were soaked in 0.5% sodium hypochlorite for 3 min. Afterward, they were rinsed three times with sterile distilled water and dried on sterilized filter paper. Based on previous studies on the cultivation conditions of pathogenic fungi, groups of four to five pieces were cultivated on a potato dextrose agar (PDA) medium, which was prepared by boiling 200 g of peeled and cubed potatoes in 1 L of water for 30 min, followed by filtering the potato extract and adding 20 g of dextrose and 15 g of agar. The prepared PDA medium was poured into plastic Petri dishes with a diameter of 9 cm for cultivation. The PDA medium was sterilized at 121 °C for 20 min before use. After sterilization, the PDA medium was poured into plastic Petri dishes when the temperature dropped slightly. After the PDA medium cooled down and solidified, the small tissue sections were placed on the PDA medium and then cultivated in the dark at 25 °C. After 4 days, the tips of the growing hyphae were subcultured onto new PDA plates for purification. Mycelia were scraped and rinsed three times with sterile water. The rinsing solution was collected and shaken thoroughly at room temperature to disperse the spore clusters. The solution was then filtered through four layers of gauze to obtain the spore suspension. The spore suspension was adjusted to 1.0 × 10⁶ spores/mL using a hemocytometer (Shanghai Qiujing Biochemical Reagent and Instrument Co., Ltd., Shanghai, China). A Water Agar (WA) medium was prepared by dissolving 5 g of agar in 500 mL of distilled water, followed by autoclaving at 121 °C for 15 min. After cooling to approximately 50 °C, the WA medium was poured into sterile Petri dishes and allowed to solidify completely. A 1 mL aliquot of the spore suspension was injected into the solidified WA medium using a sterile syringe. The plates were incubated at 25 °C for 8–10 h. Germinated single spores were identified under a Zeiss Axioscope 5 microscope (Carl Zeiss AG, Oberkochen, Germany), (400× magnification), aseptically picked with a sterile needle, and transferred to potato dextrose agar (PDA) for pure culture establishment.

2.2. Pathogenicity Testing

Healthy walnut varieties Qingxiang and Xiangling, from Longnan, Gansu Province, were washed three times with 75% ethanol and used for pathogenicity testing. To prepare the Potato Dextrose Broth (PDB) medium, 200 g of peeled and cubed potatoes were added to 1 L of distilled water, boiled for 30 min, and filtered through four layers of gauze. The filtrates were adjusted to 1 L with distilled water, mixed with 20 g of glucose, dispensed into 250 mL Erlenmeyer flasks, autoclaved at 121 °C for 20 min, and cooled to 50 °C before use. Fungal mycelia were aseptically transferred into the PDB medium using a 3 mm diameter inoculating loop sterilized by flaming in an ethanol lamp. After sealing, they were incubated in a shaker (Tianjin Ouno Instrument Co., Ltd., Tianjin, China) at 28 °C and 180 rpm for 4 days. After filtration through four layers of gauze, the spore suspension was adjusted to a final concentration of 1.0 × 10⁶ spores/mL. Using a 1 mL syringe, 100 μL of this suspension was injected into the walnut fruits. Walnut fruits injected with an equal volume of sterile distilled water were used as the control group. Each treatment included 10 walnut fruits. The inoculated walnut fruits were then maintained at 25 °C, with a 16 h/8 h light/dark cycle and 80% relative humidity. The pathogenicity test was conducted in triplicate, with symptoms observed daily and documented using a digital camera (Tokyo, Japan). Finally, fruit materials showing obvious symptoms were reisolated from the inoculated specimens to identify the pathogen species.

2.3. Observation of Morphological Characteristics

The strain was cultured on PDA plates at 25 °C in darkness. Subsequently, the fungi color and characteristics were recorded, and the size of 50 conidia was measured using an optical microscope at a magnification of 400× (Zeiss Axioscope 5, Carl Zeiss AG, Oberkochen, Germany). The conidia of the fungus cultured on a PDA plate for 14 days were collected, and the concentration of the conidial suspension was adjusted to 1.0 × 10⁶ spores/mL (following the same method as described in Section 2.2), and 20 μL of the suspension was then placed sterile hydrophobic coverslips (Dakewe (Shenzhen) Medical Equipment Co., Ltd. DKWAS001A, Shenzhen, China). The test was conducted in triplicate. In order to facilitate the formation of appressoria, the concave slides were placed in a plate to conduct incubation under the conditions of 30 °C and 2000 Lux illumination, and 3 mL sterile water was added to preserve moisture. The germination of appressoria was observed daily with an optical microscope (Zeiss Axioscope 5). The optimal time for observing spore germination was determined when the spore germination rate reaches 90% or above.

2.4. Molecular Phylogenetic Analysis

Pathogenic isolates were sub-cultured on PDA plates for 7 days at 25 °C in darkness. The mycelia were collected from the PDA plates using sterilized pipette tips. Total genomic DNA was extracted from the mycelium using the Fungi Genomic DNA Extraction Kit (Solarbio, Beijing, China) in accordance with the manufacturer’s instructions and stored at −20. Specifically, five gene regions, ITS, ACT, GAPDH, CHS-1, and TUB2, were amplified using the following primers: ITS1F/ITS4 [28], ACT-512-F/ACT-783-R and CHS-79-F/CHS-354-R [29], GDF1/GDR1 [30], and T1/βt2b [31] (

Table 1. Primers for the PCR amplification in the present study.

Region or Genes	Primer Name	Primer Sequence (5′–3′)	Annealing Temperature (°C)	References
ITS	ITS-1F ITS-4	CTT GGT CAT TTA GAG GAA GTA A TCC TCC GCT TAT TGA TAT GC	59	[28]
Act	ACT-512-F ACT-738-R	ATG TGC AAG GCC GGT TTC GC TAC GAG TCC TTC TGG CCC AT	49	[29]
GADPH	GD-F1 GD-R1	GCC GTC AAC GAC CCC TTC ATT GA GGG TGG AGT CGT ACT TGA GCATGT	56	[32]
CHS-1	CHS-79-F CHS-354-R	TGG GGC AAG GAT GCT TGG AAG AAG TGG AAG AAC CAT CTG TGA GAG TTG	60	[30]
TUB2	T1 βt2b	AAC ATG CGT GAG ATT GTA AGT ACC CTC AGT GTA GTG ACC CTT GGC	56	[31]

). The primers and PCR products utilized in this study were synthesized and sequenced by Sangon Biotech Co., Ltd. (Shanghai, China).

The PCR procedure was conducted as follows: 12.5 μL PCR Mix (2×) (Vazyme, Nanjing, China), 1 μL of each primer (10 μmol/L), 2 μL of genomic DNA (50 ng/μL), and 8.5 μL of double-distilled water were combined, resulting in a total volume of 25 μL. Five different gene regions were amplified under varying conditions. The reaction comprised 36 cycles, including an initial denaturation step at 95 °C for 5 min, followed by denaturation at 94 °C for 30 s. The optimal annealing temperatures for each gene were as follows: ACT at 59 °C, ITS region at 49 °C, CHS-1 and TUB2 at 56 °C for 30 s, and GAPDH at 60 °C for 30 s. The extension temperature was set at 72 °C for 1.5 min, with a final extension of 7 min at 72 °C performed after the cycles concluded. The PCR instrument was produced by Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The PCR products were subsequently analyzed using 1% agarose gel electrophoresis. Sequencing services were performed by Sangon Biotech Co., Ltd. (Shanghai, China).

The multilocus sequences were compared with sequences previously deposited in GenBank and the CBS database using BLAST (version 2.16.0) on NCBI, and sequences exhibiting high similarity to the queries were retrieved for phylogenetic analysis. The multilocus phylogenetic tree was constructed using MEGA 7.0, employing the maximum-likelihood method with 1000 bootstrap replications. The accession numbers of the relevant genes are saved in Table S1 of the attachment.

2.5. Biological Characteristics of the Pathogen

Mycelial plugs (5 mm diameter) from the edge of 7-day-old cultures of the isolated fungal strain Ht-10 (designated in this study) were placed on PDA plates and incubated at 25 °C. To study the effect of temperature on the growth and sporulation of Ht-10, the inoculated PDA plates were placed in the dark at 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, and 40 °C. The PDA medium was adjusted to desired pH values 5, 6, 7, 8, 9, and 10 using 1 M HCl or NaOH prior to autoclaving (Sigma Chemical Co., St. Louis, MO, USA). The mycelial plugs (5 mm diameter) of 7-day-old fungi were transferred to the center of PDA plates with different pH values and incubated at 25 °C in darkness. The light period was set as follows: full light, full dark, half-light. To study the most suitable nitrogen and carbon sources for strain Ht-10, the mycelial plugs (5 mm in diameter) were made from 7-day-old fungi and inoculated into Czapek-Dox agar (30 g/L of sucrose, 0.5 g/L of MgSO₄·7H₂O, 0.5 g/L of KCl, 3 g/L of NaNO₃, 0.01 g/L of FeSO₄, 1 g/L of KH₂PO₄, 15 g/L of agar) with different nitrogen or carbon sources while keeping other components unchanged. Soluble starch, lactose, mannitol, malt sugar, sucrose, glucose, and fructose were used to replace sucrose as the carbon source, and yeast extract, sodium nitrate, meat extracts (beef), urea, potassium nitrate, ammonium chloride, and ammonium sulfate were used to replace sodium nitrate as the nitrogen source. After being incubated in the dark in an incubator at 25 °C for 7 days, the fungus diameter was measured using the cross method, and the spore production was calculated by the hemocytometer method [33].

Test solutions for different carbon sources were prepared by dissolving 5 g each of soluble starch, lactose, mannitol, maltose, sucrose, glucose, and fructose in 500 mL of sterile water. Nitrogen source test solutions were formulated by adding 2.5 g of yeast extract, 1.0 g of sodium nitrate, 2.5 g of beef extract, 0.5 g of urea, 2.5 g of potassium nitrate, 1.0 g of ammonium chloride, and 1.0 g of ammonium sulfate to separate 500 mL of sterile water.

3. Results

3.1. Description of Symptoms in the Field

During the period from June to September of 2022, anthracnose was observed on the walnut fruits (‘Qingxiang, Xiangling’) in several walnut orchards in Longnan City, Gansu Province. At the initial stage, sub-circular or irregularly shaped spots appeared on the fruits, which were water-soaked and sunken. As the disease progressed, these spots gradually turned from brown to black, expanded, and finally coalesced into large necrotic areas (Figure 1a). A large number of gelatinous pink dots formed on the black spots of the diseased fruits under high humidity. After bringing the diseased fruits back indoors and cutting them open, they were cultured in an artificial climate chamber (Shanghai Yuejin Medical Instrument Co., Ltd. Shanghai, China) at 25 °C with a 12-h light period and high humidity. After 7 days of culture, a large number of white mycelia could be seen on the cross-section of the fruits (Figure 1c).

3.2. The Isolation of the Fungus

Fifteen isolates were obtained from the collected samples of ‘Qingxiang’ and ‘Xiangling’ walnut fruits. These isolates were classified into six types, and the representative strains of these types were named Ht-5, Ht-6, Ht-7, Ht-8, Ht-9, and Ht-10.

3.3. Pathogenicity Tests

The pathogenicity of six types of isolates was determined by the spore suspension injection method. The results showed that 3 days after inoculation, the fruits displayed slight brownness and watery lesions surrounding the wound. By the 4th day, the peels showed anthracnose symptoms characterized by brown and sunken lesions (Figure 2a). On the 7th day, the symptoms were observed to become increasingly pronounced (Figure 2b), which were similar to those initially observed in the field. On the 28th day post-inoculation, the walnut fruit appeared wrinkled, and the skin exhibited browning over a substantial area (Figure 2c). Upon cutting the fruits, severely dehydrated and wrinkled flesh was noted, accompanied by brown lesions in comparison to the control. (Figure 2d). The control fruits exhibited normal peel behavior (Figure 2e). However, no obvious symptoms appeared after inoculation with the other types of isolates.

3.4. Cultural and Morphological Characteristics

Fungi on PDA initially appeared white, gradually transitioning to light green in the center over time. The aerial mycelium was cottony, dense, and white (Figure 3a,b). The conidia were hyaline, smooth, and oblong, with a conidial anastomosis tube featuring blunt to slightly rounded ends that were unicellular. The dimensions measured 10.88–17.82 μm × 2.78–5.32 μm (n = 50) (Figure 3c). Conidia germinated and developed appressoria on hydrophobic surfaces in vitro, with most conidia forming a single appressorium. The conidial anastomosis tube varied in length, growing from the conidial ends and producing either unicellular or bicellular appressoria. Appressoria exhibited pale brown to brown coloration and were ovoid to ellipsoid in shape, with some being slightly irregular to irregularly shaped (Figure 3d–j). C. gloeosporioides and C. fructicola can be distinguished by its central orange-red coloration in later-stage colonies [34], C. acutatum by its salmon to orange conidial masses [35], and C. boninense by its longer conidia (18.5–20.0 × 7.0–9.0 μm, n = 20) [36]. Based on the results of pathogenicity tests and morphological characteristics, according to Koch’s postulates, isolate Ht-10 was determined to be the causal agent of anthracnose in walnut fruits.

3.5. Multilocus Phylogenetic Analysis

The PCR products indicated that the ITS, TUB2, ACT, CHS-1, and GAPDH genes of Ht-10 generated fragments of 500 bp, 796 bp, 274 bp, 318 bp, and 280 bp, respectively. Furthermore, based on a BLAST search, the sequence identities of the ITS (accession no. MH865005), TUB2 (accession no. JQ949943), ACT (accession no. JQ949613), CHS-1 (accession no. JQ948953), and GAPDH (accession no. JQ948622) genes of Ht-10 were 99.30%, 99.59%, 99.19%, 100%, and 99.61%, respectively, when compared to those of C. fioriniae in GenBank. A multilocus phylogenetic tree was constructed using the reference sequences (Table S1), and the results revealed that the grouping of strain Ht-10 was supported by a 100% bootstrap value (Figure 4). Based on both morphological and molecular characteristics, strain Ht-10 was identified as C. fioriniae.

3.6. Biological Characteristics of Strain Ht-10

Different conditions, including temperatures, pH, light, carbon sources, and nitrogen sources, have varying degrees of influence on the mycelial growth and sporulation of C. fioriniae. The results of the temperature test (Figure 5a) indicated that the mycelia of the pathogen could grow at 5–35 °C. Ht-10 exhibited optimal growth at 25 °C, with a colony diameter of 61.8 mm after 8 days of cultivation. The pathogen was capable of producing spores within the temperature range of 10 °C to 30 °C, with the highest spore production recorded at 30 °C, amounting to 6.4 × 10⁷ spores per 10 mL.

C. fioriniae demonstrated growth across a pH range of 5 to 10 (Figure 5b). The fungus diameter at pH 10 was the smallest among the various pH levels tested, showing significant variation across different conditions (p < 0.05), which suggests that an alkaline environment is not conducive to the pathogen’s growth. Conversely, the fungus diameter was the largest at pH 6, achieving 71.3 mm after 8 days of incubation, followed by pH 7, which yielded a diameter of 62.7 mm. C. fioriniae produced spores across the entire pH range from 5 to 10, with the highest spore production occurring at pH 6, indicating that the fungus thrives in acidic environments.

The results concerning light conditions indicated that mycelial growth was observed to be slightly enhanced under full exposure compared to the other two conditions: B (12 h of light and 12 h of darkness) and C (full darkness). After 8 days of culture, the fungus diameter reached 63.7 mm, with total spore production achieving as high as 7.8 × 10⁷ spores per 10 mL. In both full darkness and light/dark alternation environments, the fungus diameter was slower than that observed in the fully illuminated environment, and sporulation was also less pronounced. Therefore, the findings suggest that all three light conditions can be beneficial for promoting the mycelial growth and sporulation of C. fioriniae.

After inoculating on different nitrogen source media for 8 days, the mycelium of C. fioriniae was able to grow on all seven tested nitrogen sources; however, it did not produce spores under ammonium sulfate conditions (Figure 5d). Yeast extract and sodium nitrate were found to be beneficial for the fungus growth of C. fioriniae, with corresponding fungus diameters of 71.3 mm and 69.2 mm, respectively. In contrast, ammonium sulfate was the least favorable nitrogen source for mycelial growth, resulting in a fungus diameter of only 13.2 mm. When yeast extract was used as the nitrogen source, sporulation of the pathogenic fungi was most efficient, yielding a total of 8.47 × 10⁷ spores per 10 mL, which was significantly different from the results obtained with other nitrogen sources (p < 0.05). In summary, both yeast extract and sodium nitrate promote mycelial growth, while yeast extract also enhances pathogen sporulation.

After inoculating on different carbon source media for 8 days, C. fioriniae grows and produces spores with the aid of various carbon sources (Figure 5e). Notably, when soluble starch was utilized as the carbon source, it was the most conducive to the growth of the fungus compared to all the other tested carbon sources, resulting in a fungus diameter of 55.7 mm. Conversely, the fungus diameter was the smallest, reaching only 36.0 mm, when fructose was used as the carbon source. Soluble starch emerged as the most effective carbon source for spore production, yielding a total of 8.67 × 10⁷ spores per 10 mL, which was significantly different from the yields observed with other carbon sources (p < 0.05). In summary, soluble starch positively influences both mycelial growth and sporulation in pathogens.

4. Discussion

Anthracnose is a plant pathogen affecting a wide range of species across diverse geographical regions, exhibiting rapid variation and a broad host range [37]. It primarily infects bananas, strawberries, mangoes, wheat, soybeans, walnuts, and various other plants [38]. In this study, we confirmed the pathogenicity of isolated strains of walnut anthracnose using Koch’s postulates. Through morphological and molecular biological identification, we determined that the pathogen responsible for walnut anthracnose is C. fioriniae. The objectives of this research are to clarify the types of pathogenic fungi that can cause anthracnose in walnut fruit, to identify optimal culture conditions, and to provide a scientific basis for the effective control of walnut anthracnose.

Recent studies have indicated that multigene sequence analyses have been employed to elucidate the complex species within the Colletotrichum spp. [39,40], thereby addressing the limitations associated with morphological identification [41]. In this context, we utilized the ITS, ACT, GAPDH, CHS-1, and TUB2 gene regions in our research to further ascertain the taxonomic status of the strain Ht-10, which exhibited a high degree of similarity to the sequences of C. fioriniae. By integrating the findings from both morphological and multilocus molecular identifications, we ultimately confirmed the pathogen as C. fioriniae. Previous reports have documented that C. fioriniae is capable of infecting a variety of crops, including persimmons [42], apples [43], Schisandra chinensis [44], lychees [45], and grapes [39]. Moreover, Zhu et al. discovered that C. fioriniae could infect walnut leaves [46]. These research findings suggest that C. fioriniae possesses an extensive host range and significant infective capacity, thereby supporting the feasibility of this experiment.

In this experiment, it was determined that C. fioriniae can grow within a temperature range of 20 to 35 °C, consistent with the findings of Meenakshi Sharma et al. [47]. The fungus exhibits optimal growth at 25 °C and achieves maximum spore production at 30 °C. A medium with a pH of 6.0 is most conducive to both mycelial growth and spore production. The influence of varying light conditions on C. fioriniae is relatively minimal, with marginally improved mycelial growth observed under full light conditions. Yeast extract and soluble starch serve as the most suitable nitrogen and carbon sources, respectively. This finding contrasts with the biological characteristics of other reported anthracnose strains. For instance, Li et al. [48] found that C. gloeosporioides can also grow within the 20 to 35 °C temperature range, with an optimal temperature of 25 °C for both mycelial growth and spore production. Additionally, a 24-h light culture is most beneficial for both mycelial growth and spore production. Peptone and starch are identified as the most suitable nitrogen and carbon sources for mycelial growth, while yeast extract and mannitol are optimal for spore production. SONG et al. [49] determined that the optimal growth temperature for the C. fructicola strain ranges from 25 to 30 °C, with an ideal pH of 6.0 to 7.0. The most suitable nitrogen source identified is glycine, while glucose serves as the most effective carbon source. In a separate study, DAN et al. [50] found that for Colletotrichum liriopes, mycelial growth is maximized at 28 °C, and spore production peaks at 25 °C. Both mycelial growth and spore production are enhanced when the pH is maintained between 6.0 and 7.0. Additionally, full light conditions significantly promote both mycelial growth and spore production. The use of PDA notably enhances spore production, and a combination of glucose as a carbon source and yeast extract as a nitrogen source is beneficial for the mycelial growth and spore production of Colletotrichum liriopes. It is important to note that the biological characteristics of various anthracnose pathogens are not entirely uniform, which can largely be attributed to the intrinsic properties of each pathogen.

5. Conclusions

In conclusion, this study initially reported the occurrence of C. fioriniae on walnut fruits and identified the pathogen through isolation and identification, along with morphological and molecular characteristics. The most suitable conditions for growth and sporulation were screened out. This research is likely to offer valuable information for identifying and controlling the diseases caused by C. fioriniae on walnut fruits.