Study on the Identification, Biological Characteristics, and Fungicide Sensitivity of the Causal Agent of Strawberry Red Core Root Rot

: Strawberry red core root rot disease affects the growth and yield of strawberry (Fragaria ananassa Duch), which leads to economic losses in China. The study employed a tissue separation method to isolate and identify the causal agent responsible for strawberry red core root rot. This was achieved by the observation of its morphological characteristics, sequencing analyses, and pathogenicity tests. The sensitivity of five chemical fungicides against the two species of Fusarium was determined using the mycelial growth rate method, and the biological characteristics of the two species were examined. The pathogens were identified as Fusarium solani and Fusarium oxysporum . The optimal conditions for the mycelial growth of F. solani and F. oxysporum were determined to be potato sucrose agar at 25 ◦ C and pH 6, and potato dextrose agar at 30 ◦ C and pH 8, respectively, with a 24:24 light cycle. The most suitable carbon and nitrogen sources for the mycelial growth of F. solani were sucrose and sodium nitrate (NaNO 3 ), while for F. oxysporum , they were glucose and peptone. A fungicide sensitivity test indicated that Prochloraz had a good inhibitory effect on the growth of F. solani and F. oxysporum with EC50 values of 0.088 mg L − 1 and 0.162 mg. The growth inhibition effect of Azoxystrobin to F. solani and Carbendazim to F. oxysporum was not obvious. This study provides a theoretical basis for further research on strawberry red core root disease and its prevention.


Introduction
Strawberry (Fragaria ananassa Duch) is a perennial, evergreen, economically important herb in the Rosaceae family [1].Strawberries were originally planted in South America and are now widely cultivated worldwide; they are highly nutritious [2].The plants usually grow well and provide excellent quality fruit when they have sufficient light, an annual average temperature of 15-25 • C, and a soil pH of 5.8 to 6.5 [3].The common diseases of strawberry are mostly angular spot, root rot, anthracnose, and gray mold [4], which are caused by Xanthomonas fragariae [5], Fusarium spp.[6], Colletotrichum spp.[7], and Botrytis cinerea [8], respectively.
Strawberry red core root rot is a serious soilborne disease that occurs in the major areas of production for strawberry around the world [9].Hickman reported a new soilborne disease of strawberries in southern and southwestern England in 1940, which was the first report of strawberry red core root rot.A preliminary study confirmed that the causal agent of this disease was Phytophthora fragariae [10].A small-scale outbreak of strawberry red core root rot disease was also discovered in 1949 in Shizuoka City, Japan.It was confirmed that the disease was caused by Phytophthora fragariae Hickman [11].In the following decades, relevant researchers reported that a complex group of pathogens cause this disease, including Fusarium acuminatum [12], F. solani [13][14][15][16], F. oxysporum [15,17,18], and Rhizoctonia solani [18] in different areas of strawberry cultivation in northwest China and Argentina.The pathogens described above can cause the plants to grow slowly and retard their development, the leaves to wilt, and the plants to die.
The strawberry industry in Zhejiang Province, China, has developed rapidly and gradually became the primary source of strawberries for the domestic markets in China and those of other countries.The northern region of Zhejiang Province is the primary area for strawberry production in this province.However, in recent years, strawberry red core root rot has occurred frequently, which results in reduced production and a serious effect on the quality of strawberries.The broad-scale application of fungicides remains a common method of controlling crop diseases in modern agriculture [19].Currently, the primary control of strawberry red core root rot is provided by irrigation with fungicides [20].The extensive and frequent use of chemical fungicides has resulted in the development of resistance to common fungicides, such as Metalaxyl, Carbendazim, and Phenoxymeclozole [21][22][23], which has caused an excess of fungicide residues, while decreasing the control effectiveness; this seriously affects the quality of strawberries and foreign exchange earnings from exports [24].
However, there has been little research on the biological characteristics and the susceptibility of red core root rot to fungicides, except for that on pathogens that were isolated from samples of strawberry red core root in southwest and northeast China.In this study, the pathogens were identified as Fusarium solani and Fusarium oxysporum by morphological observations and molecular analyses.Moreover, the biological characteristics of the pathogen were studied to determine the best conditions for its growth.In addition, the susceptibility of the pathogens to five fungicides with different mechanisms of action was further investigated.This study aims to lay a foundation for further tests of fungicides in the field and more effective control of the pathogen.

Materials and Sample Collection
The diseased samples were collected from strawberry fields in Daochang Town, Wuxing District, Huzhou City, Zhejiang (E: 119 • 52 ′ , N: 30 • 27 ′ ) in March 2023.A total of 102 isolates separated from 56 collected samples of strawberry red column root rot were classified into two categories after morphological comparison.Compared with healthy strawberry plants, those that manifest this disease have the following characteristics: (1) The rhizome of the plant turns reddish brown in cross-section (Figure 1A); (2) the plants wither and collapse (Figure 1B), and the leaves curl and wilt (Figure 1C); and (3) the roots of diseased plants turn brown or black and have very few healthy white roots (Figure 1D).
It was confirmed that the disease was caused by Phytophthora fragariae Hickman [11].In the following decades, relevant researchers reported that a complex group of pathogens cause this disease, including Fusarium acuminatum [12], F. solani [13][14][15][16], F. oxysporum [15,17,18], and Rhizoctonia solani [18] in different areas of strawberry cultivation in northwest China and Argentina.The pathogens described above can cause the plants to grow slowly and retard their development, the leaves to wilt, and the plants to die.
The strawberry industry in Zhejiang Province, China, has developed rapidly and gradually became the primary source of strawberries for the domestic markets in China and those of other countries.The northern region of Zhejiang Province is the primary area for strawberry production in this province.However, in recent years, strawberry red core root rot has occurred frequently, which results in reduced production and a serious effect on the quality of strawberries.The broad-scale application of fungicides remains a common method of controlling crop diseases in modern agriculture [19].Currently, the primary control of strawberry red core root rot is provided by irrigation with fungicides [20].The extensive and frequent use of chemical fungicides has resulted in the development of resistance to common fungicides, such as Metalaxyl, Carbendazim, and Phenoxymeclozole [21][22][23], which has caused an excess of fungicide residues, while decreasing the control effectiveness; this seriously affects the quality of strawberries and foreign exchange earnings from exports [24].
However, there has been little research on the biological characteristics and the susceptibility of red core root rot to fungicides, except for that on pathogens that were isolated from samples of strawberry red core root in southwest and northeast China.In this study, the pathogens were identified as Fusarium solani and Fusarium oxysporum by morphological observations and molecular analyses.Moreover, the biological characteristics of the pathogen were studied to determine the best conditions for its growth.In addition, the susceptibility of the pathogens to five fungicides with different mechanisms of action was further investigated.This study aims to lay a foundation for further tests of fungicides in the field and more effective control of the pathogen.

Materials and Sample Collection
The diseased samples were collected from strawberry fields in Daochang Town, Wuxing District, Huzhou City, Zhejiang (E: 119°52′, N: 30°27′) in March 2023.A total of 102 isolates separated from 56 collected samples of strawberry red column root rot were classified into two categories after morphological comparison.Compared with healthy strawberry plants, those that manifest this disease have the following characteristics: (1) The rhizome of the plant turns reddish brown in cross-section (Figure 1A); (2) the plants wither and collapse (Figure 1B), and the leaves curl and wilt (Figure 1C); and (3) the roots of diseased plants turn brown or black and have very few healthy white roots (Figure 1D).

Isolation, Purification, and Morphological Observations
The tissue separation method [25] and the single spore separation method [26] were utilized to isolate and purify the pathogens.The fungal conidia were photographed to document their morphology, and the sizes of 30 conidiophores were measured using an optical microscope (Nikon ECLIPSE E100; Nikon, Tokyo, Japan); the color and mycelial density of the pathogenic fungi were observed on PDA [27,28].

Pathogenicity Test
The pathogenicity was verified using Koch's postulates [29].The strawberry plants were divided into two independent experimental groups, including a blank and one infected with a pathogen.Each experimental group contained 10 strawberry seedlings with three replicates.A conidial suspension (5 × 10 7 spores/mL) of the pathogen was inoculated into the roots of 10 virus-free strawberry seedlings for 10-15 min, and sterile water was used as the control.After inoculation, the plants were placed in a greenhouse at 25 • C with a relative humidity of 70% and observed daily.When these plants incurred red core root rot disease again, tissue isolation was performed on the diseased areas, and the isolated fungi were identified to determine if they were the same fungus as the inoculated fungi in order to fulfill the last step of Koch's postulates.

Morphological Characteristics
The mycelia of the pathogens were transferred to study their growth under different conditions.Fungal plugs ( = 5 mm) were placed on PDA with a cork-borer 168 h later [34,35].

Effects of Different Temperatures, pH Values, and Lighting Conditions on the Growth of Fungal Hyphae
A fungal plug was inoculated on the center of PDA in a Petri dish and incubated at 5, 10, 15, 20, 25, 30, and 35 • C.There was one plug per Petri dish ( = 90 mm) and five replicates per temperature.
Similarly, fungal plugs were tested at pH values of 4, 5, 6, 7, 8, 9, and 10, which were adjusted using HCl (1 Mol•L −1 ) and sodium hydroxide (NaOH) (1 Mol•L −1 ) under aseptic conditions, and then incubated at 25 • C.There were five replicates per pH value.Additional plates of fungal plugs were tested at full light, full darkness, and alternating light and darkness (L/D = 12 h/12 h) in a 25 • C incubator.
After 168 h of cultivation, the colony diameters were measured and recorded by the cross method under conditions of different temperatures, pH values, and lighting.

Effects of Different Carbon and Nitrogen Sources on the Growth of Fungal Hyphae
Fungal plugs were inoculated in the center of potato dextrose agar (PDA), potato sucrose agar (PSA), corn meal agar (CMA), Czapek agar (CPK), oatmeal agar (OMA), strawberry stem decoction (SSD), and water agar (WA) media and grown for 168 h in a 25 • C incubator (Table 1).Czapek agar medium was used as the basic medium, and equal amounts of glucose, D-fructose, malt dust, mannitol, and soluble starch were used to replace sucrose as the carbon source, using Czapek agar medium without a carbon source as the control.Equal amounts of peptone, glycine, beef extract, ammonium sulfate [(NH4)2SO4], and urea were used to replace sodium nitrate (NaNO 3 ) as the nitrogen source using non-nitrogen Czapek agar medium as the control.There was one plug per Petri dish ( = 90 mm) and five replicates.The colony diameters were measured after 168 h of cultivation and recorded by the cross method.

Evaluation of the Susceptibility of the Pathogens to Five Fungicides
The mycelial growth rate method [36] was used to evaluate the susceptibility of the pathogens to five fungicides (Table 2).The stock solutions of fungicides were prepared in DMSO [37] at a concentration of 10,000 µg/mL and stored at 4 • C [38].Different concentrations were added to the PDA media (Tables 3 and 4).PDA medium without fungicide was used as the control, and there were five replicates of each concentration gradient.A cork-borer was used to remove fungal plugs ( = 5 mm) from the colonies grown on PDA media without fungicides and these were placed at the center of each PDA dish that contained different concentrations of fungicides.After 168 h of cultivation, the colony diameters were measured and recorded by the cross method.
The rates of inhibition were expressed using the following equation as previously described [39]: where MCDc is the mean colony diameter of the control (without fungicide), and MCDf is the mean colony diameter for each concentration of fungicide.The toxicity regression equations were calculated with the logarithm of the concentration of the tested fungicides as X and the probability value of the inhibition rate of each treatment concentration as Y.

Statistical Analysis
SPSS 18.0 (SPSS Inc., Chicago, IL, USA) was used to calculate the mean diameter of colonies, inhibition rates, EC 50 values, correlation coefficients, and regression equations.

Statistical Analysis
SPSS 18.0 (SPSS Inc., Chicago, IL, USA) was used to calculate the mean diameter of colonies, inhibition rates, EC50 values, correlation coefficients, and regression equations.

Morphological and Molecular Identification of the Pathogens
Colonies of the F1 isolate were white, sparsely distributed, and appeared short and furry (Figure 2A).The colony edges on the bottom were white, circular, and regular (Figure 2B).The mycelia were 1.60~4.35µm wide with diaphragms spaced 7.48~28.11µm, and they were colorless (Figure 2C).Many macroconidia were visible under the microscope.They were broad, obtuse at both ends, curved inward, and of size 10.78 µm~15.42µm × 1.78 µm~2.72 µm.Most of the microconidia had 2-3 diaphragms (Figure 2D).The microconidia were mostly oval, spindle, or elliptical with amerospores and of size 3.59 µm~6.12 µm × 1.27 µm~2.55 µm with 0~1 diaphragms (Figure 2E).The pathogen was identified as Fusarium solani by morphological observation.It was seen that the colony of the F2 isolate was white, densely grown, and presented white/pink in the later stage of colonization (Figure 3A).The colony edge on the back of the PDA medium was circular and regular, appearing pink in color (Figure 3B).The mycelium was observed as solitary or clustered, light in color, often slightly bent (Figure 3C).The macroconidia of the pathogen were fewer, spindle-shaped, with a size of (28.31-31.19)µm × (5.44~5.77)µm, and 2-3 diaphragms (Figure 3D).The microconidia of the pathogen were oval or renal in shape, with a size of (8.53-18.22)µm × (3.11~4.95)µm and 0-1 diaphragm (Figure 3E).The pathogen was identified as Fusarium oxysporum, a member of Deuteromycotina, Hyphomycetes, Tuberculariales, Fusarium Link, as well.It was seen that the colony of the F2 isolate was white, densely grown, and presented white/pink in the later stage of colonization (Figure 3A).The colony edge on the back of the PDA medium was circular and regular, appearing pink in color (Figure 3B).The mycelium was observed as solitary or clustered, light in color, often slightly bent (Figure 3C).The macroconidia of the pathogen were fewer, spindle-shaped, with a size of (28.31-31.19)µm × (5.44~5.77)µm, and 2-3 diaphragms (Figure 3D).The microconidia of the pathogen were oval or renal in shape, with a size of (8.53-18.22)µm × (3.11~4.95)µm and 0-1 diaphragm (Figure 3E).The pathogen was identified as Fusarium oxysporum, a member of Deuteromycotina, Hyphomycetes, Tuberculariales, Fusarium Link, as well.
The genomic DNA of F1 and F2 was amplified by ITS1/ITS4 primers and TEF1-αF/TEF1-αR primers.There were two sequences of 522 bp and 240 bp from the F1 strain, and two sequences of 569 bp and 587 bp from the F2 strain.The sequences were submitted to the NCBI database with accession numbers PP565035/PP565036 and PP587214/PP587215 in GenBank.
The first phylogenetic tree of the F1 strain and F2 strain was constructed based on their ITS gene sequence (Figure 4A).The sequence similarity between the F1 strain and F. solani (registration number: OQ816104.1)reached 99%.The sequence similarity between the F2 strain and F. oxysporum (registration number: MH607612) reached 100%.The genomic DNA of F1 and F2 was amplified by ITS1/ITS4 primers and TEF1-αF/ TEF1-αR primers.There were two sequences of 522 bp and 240 bp from the F1 strain, and two sequences of 569 bp and 587 bp from the F2 strain.The sequences were submitted to the NCBI database with accession numbers PP565035/PP565036 and PP587214/PP587215 in GenBank.
The first phylogenetic tree of the F1 strain and F2 strain was constructed based on their ITS gene sequence (Figure 4A).The sequence similarity between the F1 strain and F. solani (registration number: OQ816104.1)reached 99%.The sequence similarity between the F2 strain and F. oxysporum (registration number: MH607612) reached 100%.
The second phylogenetic tree of the F1 strain and F2 strain was constructed based on theTEF1-α gene sequence (Figure 4B).The sequence similarity between strain F1 and F. solani (registration number: KF624788) reached 99%, while the sequence similarity between the F2 strain and F. oxysporum (registration number: ON381481) reached 100%.
The combination of these results and the morphological characteristics showed that the F1 isolate and F2 isolate from the diseased strawberry stem were Fusarium solani and Fusarium oxysporum, respectively.The phylogenetic trees based on the ITS gene sequence and the TEF1-α gene sequence were constructed as follows.

Incidence and Symptoms of F. solani and F. oxysporum
After 25-32 days of inoculation, the strawberry plants in the group that had been infected with the pathogen gradually exhibited symptoms similar to those of the disease in the field.First, the rhizome of the plant gradually exhibited a "red stele" phenomenon The second phylogenetic tree of the F1 strain and F2 strain was constructed based on the TEF1-α gene sequence (Figure 4B).The sequence similarity between strain F1 and F. solani (registration number: KF624788) reached 99%, while the sequence similarity between the F2 strain and F. oxysporum (registration number: ON381481) reached 100%.
The combination of these results and the morphological characteristics showed that the F1 isolate and F2 isolate from the diseased strawberry stem were Fusarium solani and Fusarium oxysporum, respectively.The phylogenetic trees based on the ITS gene sequence and the TEF1-α gene sequence were constructed as follows.

Incidence and Symptoms of F. solani and F. oxysporum
After 25-32 days of inoculation, the strawberry plants in the group that had been infected with the pathogen gradually exhibited symptoms similar to those of the disease in the field.First, the rhizome of the plant gradually exhibited a "red stele" phenomenon observed from the incisions (Figure 5(B4,C4)).Secondly, the strawberry roots turned black and rotted (Figure 5(B3,C3)).Third, the leaf edges curled, and the leaves withered (Figure 5(B2,C2)), and fourth, the entire strawberry plant wilted and collapsed (Figure 5(B1,C1)).The strawberry plants in the control group grew normally and did not show any symptoms of the above-mentioned diseases (Figure 5(A1-A4)), No fungi were isolated from the samples collected from the control groups.The pathogen was reisolated and cultured from diseased tissue to fulfill Koch's postulates.The morphology of the isolates obtained were consistent with those of the inoculated isolates, which proved that F. solani and F. oxysporum are the causal agents of strawberry red stele and root rot disease.3.3.Biological Characteristics of F. solani and F. oxysporum F. solani grew on all seven types of media tested and was white (Figure 6A).On PSA, the mycelia grew the most quickly at a rate of 10.38 mm d −1 .The growth rates of F. solani on PDA, CPK, OMA, and SSD were relatively similar, with values of 9.78 mm•d −1 , 10.00 mm•d −1 , 9.68 mm•d −1 , and 9.93 mm•d −1 .The mycelia grew slowly on WA media with growth rates of 5.94 mm•d −1 (Figure 6B).The mycelia grew the slowest when grown in the dark for 24 h, with values of 9.40 mm•d −1 , which was a significant difference when compared with growth in the light for 24 h and 12 h light/12 h dark (Figure 6A,C).F. oxysporum also grew on all seven media tested, and the mycelia were white (Figure 7A).They differed in their ability to grow on the different types of media tested.The mycelia grew the quickest on PDA at a rate of 12.16 mm•d −1 .They grew at 10.90 mm•d −1 and 11.11 mm•d −1 on PSA and OMA.The mycelia grew slowly on WA at 5.76 mm•d −1 (Figure 7A,B).The evaluation of light conditions showed that the mycelia grew the quickest after 24 h in the dark (11.18 mm•d −1 ), which differed significantly compared with their growth under 24 h light and 12 h light/12 h dark (Figure 7A,C).F. oxysporum also grew on all seven media tested, and the mycelia were white (Figure 7A).They differed in their ability to grow on the different types of media tested.The mycelia grew the quickest on PDA at a rate of 12.16 mm•d −1 .They grew at 10.90 mm•d −1 and 11.11 mm•d −1 on PSA and OMA.The mycelia grew slowly on WA at 5.76 mm•d −1 (Figure 7A,B).The evaluation of light conditions showed that the mycelia grew the quickest after 24 h in the dark (11.18 mm•d −1 ), which differed significantly compared with their growth under 24 h light and 12 h light/12 h dark (Figure 7A,C).
The mycelia of F. solani and F. oxysporum showed the highest growth rates at temperatures of 25 • C and 30 • C, with rates of 9.64 mm•d −1 and 12.14 mm•d −1 , respectively.The mycelial growth rates of F. solani were 6.72 and 8.42 mm•d −1 at 15 • C and 20 • C, respectively.For F. oxysporum, the growth rates were 5.08 and 8.61 mm•d −1 at the same temperatures (Figures 8A,B and 9A,B).The mycelia of F. solani grew the quickest at 9.20 mm•d −1 at pH 6 and 12.58 mm•d −1 at pH 8 (Figures 8A,C and 9A,C).F. solani could use six carbon or nitrogen sources.Its best carbon source was sucrose; on this, it grew 8.70 mm/d −1 (Figure 10A,C), which was significantly higher than its rate of growth on the other carbon sources.It grew the second best on glucose, while it had the lowest growth rate on α-lactose with 5.80 mm/d −1 .This fungus grew significantly better on NaNO 3 at 8.99 mm/d −1 (Figure 10A,B), followed by peptone, with 8.10 mm/d −1 , and urea, with 7.73 mm/d −1 , while it grew the worst on (NH4) 2 SO 4 , with 2.89 mm/d −1 .
F. oxysporum could use six carbon and nitrogen sources.It grew the best on glucose with 12.61 mm/d −1 (Figure 11A,B), which was significantly quicker than when grown on the other carbon sources.It grew the second best on malt dust, while it grew the slowest on soluble starch, with 10.72 mm/d −1 .The most effective nitrogen source for this fungus was peptone, with 12.75 mm/d −1 (Figure 11A,C).The second best was beef extract (12.54 mm/d −1 ), although there was no significant difference between the two nitrogen sources.It grew the worst on (NH4) 2 SO 4 (10.14 mm/d −1 ) and glycine (10.42 mm/d −1 ).

Effects of Five Different Fungicides on the Growth of the Pathogen
As shown in Table 3, the five tested fungicides had different inhibitory effects on the growth of F. solani, among which Prochloraz had the strongest inhibitory effect on F. solani, with an EC50 of 0.088 µg/mL, followed by Carbendazim, with an EC50 of 0.935 µg/mL, which can be used as alternative fungicides to control the disease.Among the five tested fungicides, the inhibitory effects of Azoxystrobin and Tebuconazole were relatively The small letters indicate that the difference is significant (p ≤ 0.05); +++ indicates that the mycelia grow vigorously and densely ++ indicates that the mycelia grow ordinarily, + indicates that the mycelia grow weakly and sparsely.

Effects of Five Different Fungicides on the Growth of the Pathogen
As shown in Table 3, the five tested fungicides had different inhibitory effects on the growth of F. solani, among which Prochloraz had the strongest inhibitory effect on F. solani, with an EC 50 of 0.088 µg/mL, followed by Carbendazim, with an EC 50 of 0.935 µg/mL, which can be used as alternative fungicides to control the disease.Among the five tested fungicides, the inhibitory effects of Azoxystrobin and Tebuconazole were relatively weak, while Azoxystrobin had the worst inhibitory effect on F. solani, with an EC 50 of 511.531 µg/mL, indicating that this fungicide had the lowest efficacy and was the worst at inhibiting the pathogen.Therefore, the use of Azoxystrobin as a fungicide is not recommended for further field trials with this pathogen.
As shown in Table 4, the five fungicides that were tested differed in their ability to inhibit the growth of F. oxysporum.Prochloraz was the most effective at inhibiting fungal growth, with an EC 50 of 0.162 µg/mL, followed by Tebuconazole, with an EC 50 of 0.518 µg/mL.These chemicals were effective enough to be used as alternative fungicides to control the disease.Azoxystrobin and Chlorothalonil were less effective, while Carbendazim was the least effective fungicide with an EC 50 of 20.304 µg/mL.Therefore, Carbendazim is not recommended for further field trials with this pathogen.

Discussion
Different pathogens have been reported to be the causal agents of strawberry red core root rot in China and throughout the world.Eikemo et al. [40], Chen Xiaoren et al. [41], and Chen Zhe et al. [42] all reported that the pathogen responsible for strawberry red core root rot is Phytophthora fragariae, while Mu Lisong confirmed that Fusarium solani can cause this disease.Ren Jingjing [15] found that the pathogens of strawberry red pillar root rot were Fusarium oxysporum and Fusarium solani, while Cao concluded that the types of pathogens that cause strawberry root rot may vary depending on environmental conditions, such as soil and climate [43].The pathogens of strawberry red core root rot disease were identified as Fusarium solani and Fusarium oxysporum by morphological observations and molecular techniques.This result is consistent with the findings of Ren et al. [15].No other pathogens were isolated in this study, which was primarily owing to the limited correlation of the sampling locations.The study of the biological characteristics of Fusarium oxysporum and Fusarium solani lays the foundation for in-depth research on strawberry red core root rot.
The results of this study indicate that the temperature ranges for the growth of F. solani and F. oxysporum are both 25-30 • C, which is consistent with the optimal temperatures for the growth of strawberry plants during the early stage of the occurrence of red core root rot.The optimal growth temperature for Fusarium solani is 25 • C, which is basically consistent with the findings of a study by Mu et al. [14], while the optimal growth temperature for F. oxysporum is 30 • C, which is inconsistent with that observed for the pathogen isolated from Fujian, as reported by Chen, W.L. et al. [44].The reason for this could be the influence of different hosts and climate.Both F. solani and F. oxysporum can survive in environments that range from pH 5 to 9, which indicates their high adaptability to both soil acidity and alkalinity.F. solani grows better in acidic soil environments, while F. oxysporum grows better in alkaline ones.
Different carbon and nitrogen sources have significantly different effects on the growth of F. solani and F. oxysporum, which can both utilize various carbon sources, such as sucrose, maltose, glucose, and starch, as well as various nitrogen sources, such as NaNO 3 , beef extract, and glycine.Therefore, when configuring the culture medium, attention should be paid to the selection of carbon and nitrogen sources to cultivate strains with normal development and full mycelia.
The EC 50 has been utilized as a basis to screen the degree of inhibition by fungicides [45][46][47].In this study, the sensitivity of five fungicides with different mechanisms of action on F. solani and F. oxysporum was determined.Prochloraz was the most effective among the five fungicides, which may be because it has never been used to control red core root rot on strawberry in the local area.
The sampling area and experimental results of this experiment have limitations, as they can only reflect the sensitivity of F. solani and F. oxysporum to Prochloraz in the local area and cannot indicate whether the F. solani and F. oxysporum in other strawberry red core root rot disease areas would have the same sensitivity to Prochloraz.At the same time, these results were obtained only under indoor laboratory conditions; further field experiments are needed to determine the likelihood of application of the selected fungicides on a larger scale.The short-term use of Prochloraz for the prevention and control of strawberry red core root rot can have a good effect, but if used for a long time, there may be resistance in the fungal population.Therefore, in the future, the use of Prochloraz in the field should be alternated with other effective drugs that do not show interactive resistance with Prochloraz.
This study was unable to isolate Phytophthora fragariae from the diseased samples, which may be related to the number of samples and sampling locations.The obtained research results can serve to better understand the pattern of occurrence of strawberry red core root rot in the northeastern region of Zhejiang Province and to formulate comprehensive strategies to control this disease.Further research is merited to better understand the process of pathogenicity and mechanism of infection by the pathogens.

Conclusions
This study used morphological observations, pathogenicity tests, and molecular analyses to confirm that the pathogens isolated were Fusarium solani and Fusarium oxysporum.Moreover, the biological characteristics and susceptibility of strawberry red core root rot to fungicides were determined.The most suitable conditions for the mycelial growth of F1 are PSA, 24 h of light, 25 • C, and pH 6.The most suitable carbon and nitrogen sources are sucrose and NaNO 3, respectively.In contrast, the most suitable conditions for the mycelial growth of F2 are PDA, 24 h dark, 30 • C, and pH 8.The most suitable carbon and nitrogen sources are glucose and peptone, respectively.The in vitro toxicity of five fungicides was determined and showed that Prochloraz was the most effective fungicide against both F1 and F2.This study provides a basis to prevent and control strawberry red core rot root caused by Fusarium solani and Fusarium oxysporum.

Figure 1 .
Figure 1.Red core root disease on strawberry.Note: (A) Symptoms of core root rot on strawberry stem; (B) Field status of diseased plants; (C) Status of diseased plant leaves; (D) Root status of diseased plants.

Figure 1 .
Figure 1.Red core root disease on strawberry.Note: (A) Symptoms of core root rot on strawberry stem; (B) Field status of diseased plants; (C) Status of diseased plant leaves; (D) Root status of diseased plants.

Figure 2 .
Figure 2. Morphological characteristics of F1 strain from strawberry red core root disease.Note: (A) Colony on PDA (front); (B) colony on PDA (back); (C) the morphology and size of mycelium; (D) the morphology and size of macroconidium; (E) the morphology and size of microconidium.Note: The red arrows in Figure D and Figure E indicate macroconidium and microconidium, respectively.

Figure 2 .
Figure 2. Morphological characteristics of F1 strain from strawberry red core root disease.Note: (A) Colony on PDA (front); (B) colony on PDA (back); (C) the morphology and size of mycelium; (D) the morphology and size of macroconidium; (E) the morphology and size of microconidium.Note: The red arrows in (D,E) indicate macroconidium and microconidium, respectively.

Figure 3 .
Figure 3. Morphological characteristics of the F2 strain of strawberry red core root disease.Note: (A) Colony on PDA (top); (B) colony on PDA (bottom); (C) morphology and size of the mycelia; (D) morphology and size of the macroconidia; (E) morphology and size of the microconidia.Note: The red arrows in Figure D and Figure E indicate macroconidium and microconidium, respectively.

Figure 3 . 18 Figure 4 .
Figure 3. Morphological characteristics of the F2 strain of strawberry red core root disease.Note: (A) Colony on PDA (top); (B) colony on PDA (bottom); (C) morphology and size of the mycelia; (D) morphology and size of the macroconidia; (E) morphology and size of the microconidia.Note: The red arrows in (D,E) indicate macroconidium and microconidium, respectively.Horticulturae 2024, 10, x FOR PEER REVIEW 9 of 18

Figure 4 .
Figure 4. Phylogenetic tree of the F1 stain and F2 strain based on the ITS (A) and TEF1-α (B) sequences.

18 Figure 5 .
Figure 5. Symptoms of strawberry red core root disease caused by inoculation with F1 strain and F2 strain.Note: (A1-A4): leaf parts, roots, and stem cross-sections of control group strawberry plant; (B1-B4): leaf parts, roots, and stem cross-sections of strawberry plant inoculated with F1 strain; (C1-C4): leaf parts, roots, and stem cross-sections of strawberry plant inoculated with F2 strain.3.3.Biological Characteristics of F. solani and F. oxysporum F. solani grew on all seven types of media tested and was white (Figure 6A).On PSA, the mycelia grew the most quickly at a rate of 10.38 mm d −1 .The growth rates of F. solani on PDA, CPK, OMA, and SSD were relatively similar, with values of 9.78 mm•d −1 , 10.00 mm•d −1 , 9.68 mm•d −1 , and 9.93 mm•d −1 .The mycelia grew slowly on WA media with

Horticulturae 2024 , 18 Figure 6 .
Figure 6.Effect of different media and light conditions on the mycelial growth of Fusarium solani.(A) Colony morphology when grown on different media and under different light conditions; (B) mycelial growth rate when grown on different media; (C) mycelial growth rate under different light conditions.Note: The small letters indicate that the difference is significant (p ≤ 0.05); +++ indicates that the mycelia grow vigorously and densely ++ indicates that the mycelia grow ordinarily, + indicates that the mycelia grow weakly and sparsely.

Figure 6 .
Figure 6.Effect of different media and light conditions on the mycelial growth of Fusarium solani.(A) Colony morphology when grown on different media and under different light conditions; (B) mycelial growth rate when grown on different media; (C) mycelial growth rate under different light conditions.Note: The small letters indicate that the difference is significant (p ≤ 0.05); +++ indicates that the mycelia grow vigorously and densely ++ indicates that the mycelia grow ordinarily, + indicates that the mycelia grow weakly and sparsely.

Horticulturae 2024 , 18 Figure 7 .
Figure 7. Effect of different media and light conditions on the mycelial growth of Fusarium oxysporum.(A) Colony morphology under different media and light conditions; (B) mycelial growth rate under different media; (C) mycelial growth rate under different light conditions.Note: The small letters indicate that the difference is significant (p ≤ 0.05); ++ indicates that the mycelia grow ordinarily, + indicates that the mycelia grow weakly and sparsely.

7 .
Effect of different media and light conditions on the mycelial growth of Fusarium oxysporum.(A) Colony morphology under different media and light conditions; (B) mycelial growth rate under different media; (C) mycelial growth rate under different light conditions.Note: The small letters indicate that the difference is significant (p ≤ 0.05); ++ indicates that the mycelia grow ordinarily, + indicates that the mycelia grow weakly and sparsely.

Figure 8 .
Figure 8.Effect of different temperatures and pH values on the mycelial growth of Fusarium solani.(A) Colony morphology under different temperatures and pH values; (B) mycelial growth rate under different temperatures; (C) mycelial growth rate under different pH values.Note: The small letters indicate that the difference is significant (p ≤ 0.05).

Figure 9 .
Figure 9.Effect of different temperatures and pH values on the mycelial growth of Fusarium oxysporum.(A) Colony morphology under different temperatures and pH values; (B) mycelial growth rate under different temperatures; (C) mycelial growth rate under different pH values.Note: The small letters indicate that the difference is significant (p ≤ 0.05); +++ indicates that the mycelia grow vigorously and densely ++ indicates that the mycelia grow ordinarily, + indicates that the mycelia grow weakly and sparsely.

Figure 8 . 18 Figure 8 .
Figure 8.Effect of different temperatures and pH values on the mycelial growth of Fusarium solani.(A) Colony morphology under different temperatures and pH values; (B) mycelial growth rate under different temperatures; (C) mycelial growth rate under different pH values.Note: The small letters indicate that the difference is significant (p ≤ 0.05).

Figure 9 .
Figure 9.Effect of different temperatures and pH values on the mycelial growth of Fusarium oxysporum.(A) Colony morphology under different temperatures and pH values; (B) mycelial growth rate under different temperatures; (C) mycelial growth rate under different pH values.Note: The small letters indicate that the difference is significant (p ≤ 0.05); +++ indicates that the mycelia grow vigorously and densely ++ indicates that the mycelia grow ordinarily, + indicates that the mycelia grow weakly and sparsely.

Figure 9 .
Figure 9.Effect of different temperatures and pH values on the mycelial growth of Fusarium oxysporum.(A) Colony morphology under different temperatures and pH values; (B) mycelial growth rate under different temperatures; (C) mycelial growth rate under different pH values.Note: The small letters indicate that the difference is significant (p ≤ 0.05); +++ indicates that the mycelia grow vigorously and densely ++ indicates that the mycelia grow ordinarily, + indicates that the mycelia grow weakly and sparsely.

F 1 Figure
Figure Effect of different carbon and nitrogen sources on the mycelial growth of Fusarium solani.(A) Colony morphology under different carbon and nitrogen sources; (B) mycelial growth rate under different nitrogen sources; (C) mycelial growth rate under different carbon nitrogen sources.Note: The small letters indicate that the difference is significant (p ≤ 0.05); +++ indicates that the mycelia grow vigorously and densely ++ indicates that the mycelia grow ordinarily, + indicates that the mycelia grow weakly and sparsely.

F 10 . 18 Figure 11 .
Figure 11.Effect of different carbon and nitrogen sources on the mycelial growth of Fusarium oxysporum.(A) Colony morphology under different carbon and nitrogen sources; (B) mycelial growth rate under different nitrogen sources; (C) mycelial growth rate under different carbon sources.Note: The small letters indicate that the difference is significant (p ≤ 0.05); +++ indicates that the mycelia grow vigorously and densely ++ indicates that the mycelia grow ordinarily, + indicates that the mycelia grow weakly and sparsely.

Figure 11 .
Figure 11.Effect of different carbon and nitrogen sources on the mycelial growth of Fusarium oxysporum.(A) Colony morphology under different carbon and nitrogen sources; (B) mycelial growth rate under different nitrogen sources; (C) mycelial growth rate under different carbon sources.Note:The small letters indicate that the difference is significant (p ≤ 0.05); +++ indicates that the mycelia grow vigorously and densely ++ indicates that the mycelia grow ordinarily, + indicates that the mycelia grow weakly and sparsely.

Table 1 .
Detailed components of the culture media used in this study.

Table 2 .
Detailed information of the five fungicides used in this study.

Table 3 .
Sensitivity of Fusarium solani to five different fungicides.

Table 4 .
Sensitivity of Fusarium oxysporum to five different fungicides.