First Report on Choanephora cucurbitarum Causing Choanephora Rot in Chenopodium Plants and Its Sensitivity to Fungicide

Choanephora rot of Chenopodium plants (CRC) was observed at the flowering stages in seven plantations of Shanxi Province, China. CRC had caused leaf, stem, and panicle neck rot of C. quinoa, panicle neck and stem rot of C. formosanum, and stem rot of C. album. Typical symptoms included water-soaked, rapid soft rotting, and abundant sporulation on the whole panicle necks, stems, and leaves. Based on morphological characteristics, phylogenetic analyses, and pathogenicity tests, the pathogens were identified as Choanephoraceae cucurbitarum. Sporangiola and sporangiospore of C. cucurbitarum germinated at 30 °C and were able to germinate by two h post-inoculation (hpi). The germination rates of sporangiola and sporangiospore significantly increased at 3 to 4 hpi, and the germination rates ranged from 91.53 to 97.67%. The temperature had a significant effect on the pathogenicity of C. cucurbitarum the optimum pathogenic temperatures for stems of C. quinoa, C. formosanum and C. album were 30 °C after one day post-inoculation. Choanephoraceae cucurbitarum could infect white and red quinoa panicle necks between 20 and 30 °C, and the average lesion lengths were 0.21 to 3.62 cm. Among the five tested fungicides (boscalid, dimethomorph, isopyrazam, propiconazole, and tebuconazole), isopyrazam showed higher sensitivity to sporangiola germination of C. cucurbitarum, with an EC50 value of 0.6550 μg/mL. Isopyrazam and tebuconazole strongly inhibited the sporangiospore germination of C. cucurbitarum, which showed EC50 values of 0.4406 and 0.3857 μg/mL. To our knowledge, the present study found for the first time that C. cucurbitarum is a pathogen causing panicle neck of C. formosanum and stem rot of C. formosanum and C. album, while CRC first appeared in the quinoa panicle necks, and gradually expanded to stems and leaves.

As with any crop, yield and quality may be impacted by pathogenic organisms. Among the Chenopodium plants, quinoa disease is probably the most notable. The most severe fungal diseases of quinoa included panicle rot caused by Alternaria alternata, Fusarium citri, and Trichothecium roseum [12]; gray mold caused by Botrytis cinerea [13]; stem rot and black stem caused by Choanephora cucurbitarum and Ascochyta caulina [14,15]; and leaf spot caused by Cercospora cf. chenopodii [16] and Heterosporicola beijingense [17]. Comparatively, the diseases of C. formosanum and C. album have received less attention. There is little known about the disease of C. formosanum. The diseases of C. album were mainly leaf spot caused by A. alternata [18], C. cf. chenopodii [16,19], F. equiseti [20], and Nigrospora pyriformis [21]. A. manihot Blossom blight Korea [26] Flower wet rot

Blossom blight
United States [23] Fruit soft rot Leaf blight Shoot tip dieback

Ricinus communis
Seedling rot China [22] Solanum melongena Soft rot Korea [35] Withania somnifera Leaf wet rot India [46] Stem wet rot When CRC incidences are severe, chemical control is one of the important measures. However, little research has been performed on the sensitivity of fungicides to pathogens of CRC. The toxicological effects of different types of fungicides are different, resulting in different control effects [47]. Therefore, measuring the sensitivity of pathogens to fungicides will help to control the CRC. The present study aimed to identify the species causing CRC based on morphology traits, molecular phylogenetic analysis, and pathogenicity. Our results would provide a comprehensive understanding of CRC to improve the recognition and prevention of the disease.

Sampling and Pathogen Isolation
Between July and August 2022, CRC were observed on many plantations in five regions of Shanxi Province, namely Jingle (Latitude: 38 . We collected Chenopodium plants with typical symptoms, having water-soaked and soft rot on the panicle necks, stems, and leaves. Fresh samples were the basic biological material for study. Therefore, samples were brought back to the laboratory and stored at 4 • C for further examination. Samples were randomly collected from these five counties and about 23 samples were collected. In addition, we investigated the incidence and yield loss of CRC in the field. Incidence was determined as a percentage of visual CRC symptoms on quinoas of total number of quinoas. At harvest ripeness, yield loss was estimated based on yield of diseased field and no yield loss field. Samples with monosporous sporangiola were selected and were cut into small pieces (1 × 1 cm). To obtain the pathogens, monosporous sporangiola were directly picked from the small pieces showing typical symptoms using a stereomicroscope and cultured on potato dextrose agar (PDA) (Solarbio, Beijing, China) in a climate chamber (fluorescent cycle of 12 h light/12 h dark) at 25 • C for 1 day [27,48]. Then, pure isolates were obtained using the single-mycelium tipping method on PDA and stored at 4 • C [27,49]. Morphological characteristics were used to select the representative isolates at random from all isolates for continued assessment. A total of 15 pure isolates with identical morphological characteristics were obtained, and five were randomly selected for morphology, molecular identification, and pathogenicity test.

Morphological Analysis
The representative isolates were cultured on PDA in a climate chamber (fluorescent cycle of 12 h light/12 h dark) at 25 • C for 1-3 days. The colony diameters were measured using the cross intersection method after 1 day [50]. Cultural features, including colony morphology and color, were also observed at 2 days.
The representative isolates were cultured on PDA at 25 • C for 2 days until sporangiola formed. The microscopic features of sporangiola were directly observed on PDA using an SMZ18 stereomicroscope (Nikon, Tokyo, Janpan). Microscopic structures of sporangiola were examined using a BX53 microscope (Olympus, Tokyo, Janpan) [27]. In order to view the detailed structures of sporangia and sporangiospore, the representative isolates (LMJM-2, LMJM-3, LMJM-5, LMJM-7, and LMJM-9) were, respectively, cultured on oatmeal agar (OA) (Maokang, Shanghai, China) and incubated at 25 • C for 7 days. The detailed structures of sporangia and sporangiospore were observed and measured using an SMZ18 stereomicroscope and BX53 microscope, respectively. For each representative isolate, the sizes of 50 sporangiola, sporangiophores, sporangia, and sporangiospores were randomly measured and recorded.

Molecular Identification
For DNA extraction, the representative isolates were cultured on PDA and incubated at 25 • C for 3 days. Mycelia were scraped from PDA, and then ground in liquid nitrogen. Genomic DNA was extracted using an Ezup column fungi genomic DNA purification kit (Sangon Biotech, Shanghai, China) following the manufacturer's protocol. The large subunit region (LSU) and internal transcribed spacer region (ITS) were amplified using the primer pairs LROR/LR7 and ITS1/ITS4 [51]. The PCR amplification procedures for LSU and ITS were as follows: initial denaturation at 95 • C for 5 min, followed by 35 cycles of denaturation at 95 • C for 90 s, annealing at 55 • C for 90 s, extension at 72 • C for 1 min, and a final extension at 72 • C for 10 min. The PCR amplification products were separated using 1% agarose gel electrophoresis, and the products were purified using a QIAquick Gel Extraction kit (Qiagen, Inc., Valencia, CA, USA). The PCR products were sent to Sangon Biotech (Shanghai, China) Co., Ltd. for sequencing to obtain the sequences, and uploaded to GenBank. Blakeslea trispora (CBS 564.91) was used as the outgroup for the phylogenetic tree. The maximum likelihood (ML) method was performed using PAUP (v. 4.0b10) with 1000 bootstrap replicates based on the LSU and ITS gene sequences [52]. Details of the sequences used for phylogenetic analysis are provided in Table 2. Table 2. Names, strain numbers and corresponding GenBank accession numbers of the taxa used for phylogenetic analyses. T -Ex-type strains.

Species Strain Number
GenBank Accession Number

LSU ITS
C. cucurbitarum

Sporangiola and Sporangiospore Germination
Sporangiola and sporangiospore of the representative isolates were, respectively, collected from PDA and OA. Then, spore suspensions of sporangiola and sporangiospore were, respectively, prepared at a concentration (1 × 10 5 cfu/mL) with sterile distilled water. The PDA temperature was at~50 • C, and 200 µL of the PDA was applied to the sterile microscope slides (26 × 76 mm) [15]. After PDA solidification, the suspension (20 µL) was inoculated on the ready-prepared microscope slide and incubated in a desiccator with a relative humidity (RH) of 75% (saturated NaCl saline solution) at 30 • C. After 1, 2, 3, and 4 h, the morphologies of sporangiola and sporangiospore germination were observed using a BX53 microscope, and counted to determine the germination rate from 200 spores in each of the three replicates.

Pathogenicity Tests
The pathogenicity of all representative isolates (LMJM-2, LMJM-3, LMJM-5, LMJM-7, and LMJM-9) was assessed on healthy plants of Chenopodium quinoa, C. formosanum, and C. album. Chenopodium quinoa (white quinoa: Jingli No. 1, red quinoa: Jingli No. 3), C. formosanum (Xinli No. 1), and C. album were cultivated in the greenhouse from seeds until the flowering stage (fluorescent cycle of 12 h light/12 h dark). To determine the pathogenicity of sporangiola on the panicle necks of C. quinoa and C. formosanum, the panicle necks were rinsed with sterile distilled water several times and then air-dried. Then, the sterile cotton wools were immersed in the prepared sporangiola suspension (~200 µL, 1 × 10 5 cfu/mL) and inoculated on panicle necks [27]. The representative isolates were inoculated on 5 plants (one plant per pot). The control plants were treated in the same way with sterile distilled water. Each treatment was administered 3 times and conducted twice. Before inoculation stems of C. quinoa, C. formosanum, and C. album, the stems were rinsed with sterile distilled water several times and then air-dried. Sporangiola suspension (1 × 10 5 cfu/mL) was inoculated on stems, as previously described. The representative isolates were inoculated on 5 plants. The control plants were inoculated in the same way with sterile distilled water. Each treatment consisted of three replicates and the experiment was conducted twice. In addition, the quinoa leaves were surface sterilized, and 0.5 mL of the sporangiola suspension (1 × 10 5 cfu/mL) was inoculated on the leaf surface using a sterile handheld sprayer. Control leaves were inoculated in parallel using sterile distilled water. Each treatment was applied to three leaves and repeated five times. After inoculation, all inoculated and control plants described above were incubated in a climate chamber at 30 • C and RH = 75 ± 2%, with a 12 h photoperiod. The symptoms were monitored and recorded over 1-3 days, until the experiments were completed.
To measure the effect of temperature on infection, we used a sporangiola suspension (1 × 10 5 cfu/mL) inoculated on the stems of C. quinoa, C. formosanum, and C. album (10 plants per replicate) and panicle necks of white and red quinoa (10 plants per replicate). The control group was inoculated similarly with sterile distilled water. The experiment was conducted twice, and each treatment consisted of three replicates. After inoculation, all inoculated and control plants were placed in a climate chamber (RH = 75 ± 2%, 12 h photoperiod) with a temperature gradient of 10, 15, 20, 25, and 30 • C. The lesion lengths on the stem and panicle neck were measured after 3 days post-inoculation (dpi). To confirm Koch's postulates, pathogens were reisolated and reidentified from symptomatic panicle necks, stems, and leaves of all inoculated plants.

Sensitivity of Sporangiola and Sporangiospore Germination to Five Fungicides
In order to identify the inhibition activity of fungicides on the germination of spores (sporangiola and sporangiospores) of C. cucurbitarum, we screened 5 fungicides. Boscalid (97.0%), dimethomorph (98.5%), isopyrazam (95.0%), propiconazole (95.4%), and tebuconazole (97.3%) were, respectively, dissolved in acetone to prepare 1 × 10 4 µg/mL stock solutions [47,53]. The stock solutions of five fungicides were diluted into serial dilutions using sterile distilled water and added to PDA at~50 • C to prepare the fungicide-containing PDAs [15,53] (Table 3). Preliminary testing showed that acetone was less than 0.25%; this did not affect the sporangiola and sporangiospore germination. Therefore, the same volume of acetone was added to PDA as a blank control. Different serial dilutions of the fungicide-containing PDAs were prepared (Table 3); 200 µL of each of the fungicide-containing PDAs was applied to the sterile microscope slides (26 × 76 mm) [15]. Spore suspension (20 µL, 1 × 10 5 cfu/mL) was inoculated onto the ready-prepared microscope slide after agar solidification and incubated in a desiccator at 25 • C and RH = 75%. Spore germination was, respectively, counted to determine the germination inhibition rates after 4 h [15,53]. The experiment was performed twice, and each fungicide treatment and control contained three replicates.
The log transformation of the each treatment fungicide concentration represented the independent variable (X) and the probability of the corresponding germination inhibition rate represented the dependent variable (Y). With the regression equation, the EC 50 value with a 95% confidence level to each treatment fungicide was determined [47,54].

Data Statistics and Analysis
Data were analyzed with SPSS statistics 19.0 by one-way ANOVA, and means were compared using Tukey's test at a significance level of p = 0.05. Letters indicate significant differences (p = 0.05).

Field Symptoms
Choanephora rot of Chenopodium plants (CRC) primarily infected panicle necks, stems, and leaves. The incidence of CRC was approximately 65%, and the yield of C. quinoa and C. formosanum might decrease by over 80% in the fields where diseases were the most severe in Jingle, Taigu, and Xinzhou of Shanxi Province. These diseased panicle necks, stems, and leaves were usually discoloured, water-soaked, and soft rotted ( Figure 1). Interestingly, CRC first infected the quinoa panicle necks, and then gradually spread towards to the stems and leaves ( Figure 1A). The initial symptoms began as pale to tan lesions, and the margins between the lesions and healthy tissues were clear ( Figure 1A). Subsequently, the color of the lesions on quinoa turned brown to black and water-soaked, resulting in rapid soft rotting of the whole panicle necks ( Figure 1A). In the later stages, abundant sporulation occurred along the panicle necks, and then encompassed the entire panicles, resulting in quinoa panicles being blighted ( Figure 1A). When CRC infected the quinoa stems, it primarily appeared in the middle and lower branches of the main stems ( Figure 1A). Symptoms on the quinoa stems consisted of brown to black coloring and water-soaking, and followed by rapid soft rot ( Figure 1A). Symptoms on quinoa leaves first developed on petioles resulting in wilting and rotting, and then expanded to leaves. Initial symptoms on the base of leaves appeared as water-soaked and darkgreen. A soft rot developed together with abundant sporulation and led to quinoa leaves' blight ( Figure 1A).
( Figure 1A). Symptoms on the quinoa stems consisted of brown to black coloring and water-soaking, and followed by rapid soft rot ( Figure 1A). Symptoms on quinoa leaves first developed on petioles resulting in wilting and rotting, and then expanded to leaves. Initial symptoms on the base of leaves appeared as water-soaked and darkgreen. A soft rot developed together with abundant sporulation and led to quinoa leaves' blight ( Figure 1A).
Additionally, CRC primarily infected the panicle necks and stems of C. formosanum, and did not usually infect the leaves ( Figure 1B). Stems symptoms on C. formosanum appeared pale to grayish, with necrotic lesions, and they were covered with masses of sporangiola ( Figure 1B). In contrast, stem symptoms on C. album initially consisted of pale to tan necrotic lesions, resulting in the infected stems breaking off the rest of the plant ( Figure  1C). Additionally, CRC primarily infected the panicle necks and stems of C. formosanum, and did not usually infect the leaves ( Figure 1B). Stems symptoms on C. formosanum appeared pale to grayish, with necrotic lesions, and they were covered with masses of sporangiola ( Figure 1B). In contrast, stem symptoms on C. album initially consisted of pale to tan necrotic lesions, resulting in the infected stems breaking off the rest of the plant ( Figure 1C).

Morphological Characteristiscs of the Choanephora cucurbitarum
The colonies grew rapidly on PDA, reaching 74-76 mm diameters in one day. After two days, colonies were white and cottony, with scattered monosporous sporangiola, and appearing pale yellow from below ( Figure 2A).
A phylogenetic tree was constructed using Blakeslea trispora (CBS 564.91 T ) as the outgroup. The results showed that the representative isolates (LMJM-2, LMJM-3, LMJM-5, LMJM-7, and LMJM-9) clustered in the same branch as sixteen isolates of C. cucurbitarum ) with a 98% bootstrap support rate, indicating that the representative isolates were the closest relationship with C. cucurbitarum (Figure 3).

Sporangiola and Sporangiospore Germination of Choanephora cucurbitarum
Sporangiola and sporangiospores of C. cucurbitarum were germinated at 30 • C, and the morphology of sporangiola and sporangiospore germination were separated at two representative stages of germ tubes formation and germ tubes elongation. At the stage of germ tubes formation, the germ tubes were able to germinate from the central part of the sporangiola and sporangiospore by 2 h post-inoculation (hpi) (Figure 4A,B). The mean germ tube lengths of sporangiola and sporangiospores were 11.77 and 8.95 µm. At the stage of germ tubes elongation, the branches of germ tubes appeared and the mean germ tube lengths of sporangiola and sporangiospores were 23.90-39.26 µm and 25.62-54.13 µm by 3-4 hpi. Germination rates of sporangiola and sporangiospores were 77.43% and 70.67% at 2 hpi. The germination rates of sporangiola and sporangiospores significantly increased at 3-4 hpi, and the germination rates ranged from 91.53 to 97.67%, and the differences were not significant ( Figure 4C).  A phylogenetic tree was constructed using Blakeslea trispora (CBS 564.91 ) as the out-group. The results showed that the representative isolates (LMJM-2, LMJM-3, LMJM-5, LMJM-7, and LMJM-9) clustered in the same branch as sixteen isolates of C. cucurbitarum (KUS-F27538, KUS-F27657, KUS-F27485, KUS-F28066, CBS 674.93, KUS-F27540, KUS-F28029, KUS-F29113, JSAFC2347, KA47639, KA47637, QJFY1, JSAFC2346, JSAFC2348, CBS 178.76 T , and JPC1) with a 98% bootstrap support rate, indicating that the representative isolates were the closest relationship with C. cucurbitarum (Figure 3).

Sporangiola and Sporangiospore Germination of Choanephora cucurbitarum
Sporangiola and sporangiospores of C. cucurbitarum were germinated at 30 °C, and the morphology of sporangiola and sporangiospore germination were separated at two representative stages of germ tubes formation and germ tubes elongation. At the stage of germ tubes formation, the germ tubes were able to germinate from the central part of the sporangiola and sporangiospore by 2 h post-inoculation (hpi) ( Figure 4A,B). The mean germ tube lengths of sporangiola and sporangiospores were 11.77 and 8.95 µm. At the stage of germ tubes elongation, the branches of germ tubes appeared and the mean germ tube lengths of sporangiola and sporangiospores were 23  Pathogenicity tests showed that C. cucurbitarum could infect quinoa panicle necks, stems, and leaves. No symptoms were observed in the control groups ( Figure 5A). One day after inoculation, pale brown necrosis lesions were found at the inoculation sites of white quinoa panicle necks, and the margins between the lesions and healthy tissues were obvious. By comparison, the inoculation red quinoa panicle necks were greyish-white, water-soaked, and soft rotted. At 2 dpi, the lesions further enlarged, and the lesions' lengths ranged from 5.71 to 6.29 cm. Noticeably, the lesions on white quinoa were brown to black, water-soaked, and soft rotted. At 3 dpi, obvious CRC symptoms that were identical to the naturally infected panicle necks were observed ( Figure 5A). The color of the lesions on white and red quinoa stems induced by C. cucurbitarum were different. At 1 dpi, obvious black and water-soaked lesions were found on the white quinoa stems; however, pale brown necrosis lesions were found on the red quinoa stems. With the development of disease, the typical symptoms developed on inoculated stems and were covered with masses of sporangiola at 3 dpi ( Figure 5A). In addition, pathogenicity tests of C. cucurbitarum were performed on the quinoa leaves. At 1 dpi, obvious black and water-soaked lesions were found on the quinoa petioles and leaves. The color of the diseased leaves gradually became dark green, with a film of mold, resulting in rapid soft rotting of the whole ( Figure 5A). There were no symptoms in the control ( Figure 5A).
The pathogenicity tests of C. cucurbitarum were further inoculated on the panicle necks and stems of C. formosanum and stems of C. album. Three days after inoculation, obvious symptoms appeared on the panicle necks and stems of C. formosanum and stems of C. album, the inoculated panicle necks and stems were covered with masses of sporangiola. The control plants of C. formosanum and C. album remained healthy ( Figure 5B,C). C. cucurbitarum was reisolated from the panicle necks, stems, and leaves that showed symptoms, and their reidentification was confirmed by morphology and molecular characterizations, as described above. Collectively, the morphology, molecular characterization, and pathogenicity confirmed that C. cucurbitarum was the causal agent of CRC.
J. Fungi 2023, 9, x FOR PEER REVIEW 11 of 19 increased at 3-4 hpi, and the germination rates ranged from 91.53 to 97.67%, and the differences were not significant ( Figure 4C).

Effect of Temperature on the Pathogenicity of Choanephora cucurbitarum
Temperature had a significant effect on the pathogenicity of C. cucurbitarum ( Figure 6). Choanephora cucurbitarum could infect the stems of C. quinoa, C. formosanum, and C. album between 20 and 30 • C. The optimum pathogenic temperature for stems of C. quinoa, C. formosanum, and C. album was 30 • C, and the lesions lengths were 8.93, 7.10, and 1.22 cm, respectively. When the temperatures were below 15 • C, there were no lesions in all stems ( Figure 6A).

Effect of Temperature on the Pathogenicity of Choanephora cucurbitarum
Temperature had a significant effect on the pathogenicity of C. cucurbitarum ( Figure  6). Choanephora cucurbitarum could infect the stems of C. quinoa, C. formosanum, and C. album between 20 and 30 °C. The optimum pathogenic temperature for stems of C. quinoa, C. formosanum, and C. album was 30 °C, and the lesions lengths were 8.93, 7.10, and 1.22 cm, respectively. When the temperatures were below 15 °C, there were no lesions in all stems ( Figure 6A).
Choanephora cucurbitarum could infect white and red quinoa panicle necks between 20 and 30 °C, and the average lesions lengths were 0.21-3.62 cm. The optimal pathogenic temperature of C. cucurbitarum was 30 °C, and the lesions lengths were 1.76 cm and 3.62 cm, which were significantly higher than other treatments. When the temperature was at 20 °C, the lesions lengths were significantly reduced to 0.21 cm on white quinoa and 0.33 cm on red quinoa. At 10 and 15 °C, the lesions lengths were 0 cm ( Figure 6B). Figure 6. Effect of temperature on pathogenicity of the representative isolates of Choanephora cucurbitarum. (A) Pathogenicity on stems of C. quinoa, C. formosanum, and C. album at different temperatures, (B) lesions lengths on panicle necks of white and red quinoa that were inoculated with Choanephora cucurbitarum and incubated at different temperatures. Data were analyzed with SPSS statistics 19.0 by one-way ANOVA, and means were compared using Tukey's test at a significance level of p = 0.05. Different letters indicate significant differences (p = 0.05).

Effect of Five Fungicides on Spore Germination of Isolate LMJM-2
The spores (sporangiola and sporangiospore) of C. cucurbitarum showed different sensitivity to five fungicides. Isopyrazam was found to be the most effective fungicide against sporangiola germination of C. cucurbitarum, with an EC50 value of 0.6550 µg/mL, and the differences compared with the other four fungicides were significant. Furthermore, the EC50 values of boscalid, dimethomorph, propiconazole, and tebuconazole were   Choanephora cucurbitarum could infect white and red quinoa panicle necks between 20 and 30 • C, and the average lesions lengths were 0.21-3.62 cm. The optimal pathogenic temperature of C. cucurbitarum was 30 • C, and the lesions lengths were 1.76 cm and 3.62 cm, which were significantly higher than other treatments. When the temperature was at 20 • C, the lesions lengths were significantly reduced to 0.21 cm on white quinoa and 0.33 cm on red quinoa. At 10 and 15 • C, the lesions lengths were 0 cm ( Figure 6B).

Effect of Five Fungicides on Spore Germination of Isolate LMJM-2
The spores (sporangiola and sporangiospore) of C. cucurbitarum showed different sensitivity to five fungicides. Isopyrazam was found to be the most effective fungicide against sporangiola germination of C. cucurbitarum, with an EC 50 value of 0.6550 µg/mL, and the differences compared with the other four fungicides were significant. Furthermore, the EC 50 values of boscalid, dimethomorph, propiconazole, and tebuconazole were 29.1273, 16.7763, 28.6449, and 4.1957 µg/mL, respectively ( Figure 7A). Among the five fungicides, those that most strongly inhibited the sporangiospore germination of C. cucurbitarum were isopyrazam and tebuconazole, which showed EC 50 values of 0.4406 and 0.3857 µg/mL. The differences between isopyrazam and tebuconazole were not significant, but the differences compared with the other three fungicides were significant. Moderate inhibitory effects on the sporangiospore germination were boscalid and dimethomorph, which showed EC 50 values of 1.0250 and 1.3493 µg/mL. In contrast, propiconazole showed a low inhibitory effect against the sporangiospore of C. cucurbitarum, with an EC 50 value of 12.4997 µg/mL ( Figure 7A). tarum were isopyrazam and tebuconazole, which showed EC50 values of 0.4406 and 0.3857 µg/mL. The differences between isopyrazam and tebuconazole were not significant, but the differences compared with the other three fungicides were significant. Moderate inhibitory effects on the sporangiospore germination were boscalid and dimethomorph, which showed EC50 values of 1.0250 and 1.3493 µg/mL. In contrast, propiconazole showed a low inhibitory effect against the sporangiospore of C. cucurbitarum, with an EC50 value of 12.4997 µg/mL ( Figure 7A).
As in the control treatment, when cultured on PDA-containing fungicide, the sporangiola and sporangiospore germinations of C. cucurbitarum were normal. The germ tubes emerged from the central part of the sporangiola and sporangiospore, and the shape of the germ tubes was normal ( Figure 7B). Mean germ tube lengths of sporangiola/sporangiospore were shorter than those at the control treatment, which were 5.12/8.45, 3.91/7. 37, 9.32/7.54, 7.18/7.23, and 6.37/6.53 µm on PDA containing boscalid, dimethomorph, isopyrazam, propiconazole, and tebuconazole, respectively. On PDA without any fungicide, the germ tube lengths of sporangiola and sporangiospore were 48.29 and 45.65 µm ( Figure  7B).

Discussion
Choanephora cucurbitarum was frequently associated with rot on the flower, stem, and leaf of a variety of hosts (Table 1). In the past, Sun et al. also reported that on quinoa stem rot caused by C. cucurbitarum in China [14]. The present study showed for the first time that C. cucurbitarum could cause the rot of quinoa panicle neck and leaf in China, which could lead to a decrease in yield. Our research indicated that CRC on quinoa first appeared in the panicle neck, and gradually expanded to the stem and leaf. Similarly, findings suggested that C. cucurbitarum mostly infected flowers and young fruits [24,30]. Therefore, it As in the control treatment, when cultured on PDA-containing fungicide, the sporangiola and sporangiospore germinations of C. cucurbitarum were normal. The germ tubes emerged from the central part of the sporangiola and sporangiospore, and the shape of the germ tubes was normal ( Figure 7B). Mean germ tube lengths of sporangiola/sporangiospore were shorter than those at the control treatment, which were 5.12/8.45, 3.91/7.37, 9.32/7.54, 7.18/7.23, and 6.37/6.53 µm on PDA containing boscalid, dimethomorph, isopyrazam, propiconazole, and tebuconazole, respectively. On PDA without any fungicide, the germ tube lengths of sporangiola and sporangiospore were 48.29 and 45.65 µm ( Figure 7B).

Discussion
Choanephora cucurbitarum was frequently associated with rot on the flower, stem, and leaf of a variety of hosts (Table 1). In the past, Sun et al. also reported that on quinoa stem rot caused by C. cucurbitarum in China [14]. The present study showed for the first time that C. cucurbitarum could cause the rot of quinoa panicle neck and leaf in China, which could lead to a decrease in yield. Our research indicated that CRC on quinoa first appeared in the panicle neck, and gradually expanded to the stem and leaf. Similarly, findings suggested that C. cucurbitarum mostly infected flowers and young fruits [24,30]. Therefore, it is necessary to monitor quinoa panicle neck rot in the field. Quinoa panicle neck rot is the early stage of CRC and also the critical period of disease management. C. cucurbitarum mainly infected quinoa panicle neck and stem but could also cause quinoa leaf rot in China. Compared with the leaf rot of other hosts, C. cucurbitarum can result in rapid soft rotting of the whole quinoa leaves (Table 1). These results suggest that the infection of quinoa leaves should raise concern and further investigation.
Currently, C. formosanum is grown as an ornamental and grain crop and C. album is a native weed in China [55]. For the time being, there is no report about the association of C. cucurbitarum on the panicle neck and stem of C. formosanum and C. album. The present study found for the first time that C. cucurbitarum is the pathogen causing panicle neck and stem rot of C. formosanum and stem rot of C. album. It should be noted that CRC symptoms on C. formosanum and C. album growing adjacent to infected quinoa were observed. We speculate that the host ranges of C. cucurbitarum have extended and are likely to continue expanding. Therefore, it is important to reduce further spread of CRC. The proper layout C. formosanum and C. quinoa and removal C. album in the field are essential for control of CRC. Interestingly, CRC is not observed in the panicle necks of C. album. It is hypothesized that panicle traits are also probably one of the factors. The lax panicles of C. album may keep them in relatively low humidity that is not infected by CRC, especially during periods of rainfall.
Correct diagnosis is a fundamental requirement for effective disease management. As shown in previous studies, the three genes (LSU, ITS, and SSU) for identification of the genus of Choanephora have been used for resolution at the species level [27,31,56]. We found that a lot of reference strains were from the CBS culture collection whose SSU were unknown [56]. Because the reference strains had only one or two sequences of the three genes, the phylogenetic tree would be different. Therefore, both ITS and LSU are recommended as the most useful genes for the identification of Choanephora species. In future, more phylogenetically informative genes are required to identify the genus of Choanephora, especially such as SSU. In this study, the representative isolates (LMJM-2, LMJM-3, LMJM-5, LMJM-7, and LMJM-9) clustered in the same branch as C. cucurbitarum based on the analysis of LSU and ITS. The morphological characterization (sporangiola, sporangia, and sporangiospores) of the representative isolates that infected Chenopodium plants were generally consistent with the model strain of C. cucurbitarum. A set of morphology, molecular characterization, and pathogenicity evaluation identified C. cucurbitarum as the pathogen causing CRC.
The Choanephora diseases frequently occur in tropical and subtropical regions featuring high temperatures and humidity [57]. Our results showed that the developments of CRC were very rapid, with a very short time (1 to 3 days). The germination rates of the sporangiola and sporangiospores of C. cucurbitarum were 77.43% and 70.67% by 2 hpi at 30 • C and RH = 75%. Pathogenicity tests showed that C. cucurbitarum could infect the panicle necks of C. quinoa and C. formosanum, and the stems of C. quinoa, C. formosanum, and C. album at 30 • C after 1 dpi. This may also partially explain why CRC outbreaks could appear within a very short time, particularly during high humidity and temperatures. Is has also been reported that the suitable environmental conditions (25 to 30 • C and 70-90% relative humidity) could promote infection of C. cucurbitarum [28,31,57,58]. Between July and August in 2022, the weather conditions of C. formosanum and C. quinoa plantations of Shanxi Province were hot and humid, which were preferred by C. cucurbitarum for infections. This also can explain why Chenopodium plants are more susceptible to CRC in summer. Noteworthily, there are two kinds of sporangiola and sporangiospores in C. cucurbitarum, which indicates the need for targeted control.
Sporangiola and sporangiospores of C. cucurbitarum play an important role in early infection, resulting in a rapid spread and devastating loss of CRC. Inhibition germination of sporangiola and sporangiospores is important in the early prevention of CRC. The toxicological effects of the same types of fungicide can differ in the same pathogen [47].
Among the five fungicides in this study, isopyrazam had the strongest inhibitory effects on sporangiola germination. Meanwhile, isopyrazam and tebuconazole had relatively high inhibitory effects against sporangiospore germination. We speculate that isopyrazam and tebuconazole may provide preventive activities to control CRC. Tebuconazole and propiconazole are both triazole fungicides, but the EC 50 s of tebuconazole and propiconazole to C. cucurbitarum are different. This may be related to the molecular structure of tebuconazole and propiconazole and may also be related to the targets of tebuconazole and propiconazole in C. cucurbitarum. Additionally, boscalid and dimethomorph have moderate inhibitory activities against sporangiospore germination. This demonstrates the potential of boscalid and dimethomorph to combat CRC.

Conclusions
In conclusion, this firstly reports the occurrence of C. cucurbitarum on Chenopodium plants based on morphological characteristics, phylogenetic analysis, and pathogenicity analysis in many regions of Shanxi, China. Among the five tested fungicides, isopyrazam showed a higher sensitivity to sporangiola germination of C. cucurbitarum. Isopyrazam and tebuconazole strongly inhibited the sporangiospore germination of C. cucurbitarum. Therefore, isopyrazam and tebuconazole may provide preventive activities to control CRC. The findings of this study will provide important information on the recognition, diagnosis, and management of these diseases. In the future, surveys of this pathogen are needed to assess its genetic diversity, infection mechanisms, and epidemiology to combat it. Additionally, different strains of C. cucurbitarum from different countries and research on the pathogenicity and host range between different strains need the cooperation of researchers.