First Report of Black Spot Disease Caused by Alternaria alternata on Persimmon Fruit in China.

Sweet persimmon is native to Japan and valued for its fruit, which are high in sugar and vitamins. In October 2021, symptoms were observed on persimmon (Diospyros kaki L. cv. Yangfeng) fruits in cold storage room in Suiping county, Henan Province (32.59 °N, 15 113.37 °E). Initially, small circular dark-brown spots were visible on the fruit rind, turning into irregular sunken dark areas, and eventually rotting 15% of 200 fruits after four weeks of cold storage (10°C, 95% relative humidity). To isolate the causal agent, 10 fruits of symptomatic tissues (4 mm2) were surface-sterilized in 2% sodium hypochlorite (NaOCl) for 1 minute, washed three times in sterile distilled water, then aseptically transferred to potato dextrose agar (PDA) and incubated for 7 days at 25°C. Fungal colonies were isolated from plant tissue, and on three colonies of similar morphology, single-spore isolation was performed. On PDA, the isolates produced circular colonies of fluffy aerial mycelia, gray-brown in the center with gray-white margins. Conidia were dark brown, obclavate or pyriform, with 0 to 3 longitudinal septa and 1 to 5 transverse septa, and a size range of 19.2 - 35.1 × 7.9 - 14.6 µm (n=100). Conidiophores were olivaceous, septate, straight, or bent, with a length of 18 - 60 × 1 - 3 µm (n=100). These morphological characteristics identify the isolates as Alternaria alternata (Simmons. 2007). Genomic DNA was extracted from a representative isolate YX and re-isolated strain Re-YX by cetyltrimethylammonium bromide (CTAB). The primers of ITS1/4, Alt-F/R, GPD-F/R, EF1/2, EPG-F/R (Chen et al. 2022), RPB2-5F/7cR (Liu et al. 1999), and H3-1a/1b (Lousie et al. 1995) were used to amplify the partial internal transcribed spacer (ITS) region, Alternaria major allergen (Alt a1), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF), endo-polygalacturonase (endoPG), RNA polymerase second largest subunit (RPB2) and Histone 3 (His3), respectively. GenBank accession No of ITS, Alt a1, GAPDH, TEF, endoPG, RPB2, His3 were ON182066, ON160008 to ON160013 for YX and OP559163, OP575313 to OP575318 for Re-YX respectively. Sequence data of Alternaria spp. were downloaded from GenBank and the BLAST analysis showed 99%-100% homology between various A. alternata strains (ITS: MT498268; Alt a1: MF381763; GAPDH: KY814638; TEF: MW981281; endoPG: KJ146866; RPB2: MN649031; His3: MH824346). A phylogenetic analysis based on ITS, Alt a1, GAPDH, TEF, and RPB2 sequences using MEGA7 (Molecular Evolutionary Genetics Analysis) revealed that the isolate YX and Re-YX were clustered in A. alternata clade (Demers M. 2022). For the pathogenicity test, seven-day-old cultures were used to create spores suspensions (5.0 × 105 spores/mL) of each of the three isolates. Ten µL aliquots from each isolate were inoculated onto ten needle-wounded persimmon fruits; ten additional fruits were inoculated with water only to serve as controls. The pathogenicity test was three replications. Fruits were deposited in a climate box at 25°C, 95% relative humidity. Seven days post-inoculation, the wounded fruit treated with spore suspensions displayed black spot symptoms similar to the symptoms on the original fruit. There were no symptoms on the control fruits. The strain Re-YX was re-isolated from the symptomatic tissue of inoculated fruits and its identity confirmed using the morphological and molecular methods previously mentioned, fulfilling Koch's postulates. The persimmon fruit rot caused by A. alternata had been reported in Turkey and Spain (Kurt et al., 2010, Palou et al., 2012). According to our knowledge, this is the first report of black spot disease on persimmon fruits caused by A. alternata in China. The disease could infect persimmon fruits during cold storage, so more control methods should be developed to prevent postharvest disease of persimmon in the future.

First Report of Fruit Rot Caused by Fusarium graminearum on Ponkan (Citrus reticulata Blanco cv. Ponkan) in China.

Ponkan (Citrus reticulata Blanco cv. Ponkan) is a Chinese citrus species with tasty fruit. In November 2021, an unknown postharvest disease of Ponkan fruit caused nearly 15% losses of 2000 fruits in Nanchang, Jiangxi Province (28.68° N, 115.85° E). The initial fruit's surface necrosis was brown (Xu et al. 2022) (Figure 1A). Disease spots spread to the entire fruit, white or grey hyphae appeared, and the fruit rotted. Twenty diseased fruits were surface-disinfested with 2% sodium hypochlorite and 75% ethanol, then rinsed with sterile distilled water to isolate the pathogen. Diseased tissue sections (5 × 3 mm) were incubated on potato dextrose agar (PDA) for 7 days at 25°C. Twelve of 15 monoconidial isolates have similar morphology. On PDA, the isolates produced copious white aerial mycelia. After 5-7 days on straw juice medium, two types of conidia appeared (Rice straw 60 g, Agar 20 g, distilled water 1000 mL) (Figure 1E-I). Macroconidia were abundant, falcate, slender, and slightly curved with 0-8 septa, mostly 4-5 septa (average 41.70 × 3.81 m, n=100) (Figure 1J). Microconidia were globose, oval, or piriform with 0-1 septa, 2.72 to 8.57 × 2.53 to 7.47 m (average 5.49 × 4.52 m, n=50) (Figure 1L), and chlamydospores were not observed. Conidial and colony morphology identified 12 monoconidial isolates as Fusarium graminearum (Fisher et al., 1982; Yulfo-Soto et al., 2021). Genomic DNA was extracted from three isolates using a DNA Extraction Kit (Yeasen, Shanghai, China). The ITS1/4 region combined with partial gene fragments of translation elongation factor-1 alpha (TEF-1α, primer TEF1/2, O'Donnell et al. 1998), RNA polymerase second largest subunit (RPB2, primer fRPB2-5F/7cR, Liu et al. 1999) and ß-tubulin (ß-tub, primer Bt2a/2b, Li et al. 2013) from the isolates were amplified and sequenced. The three tested isolates showed identical gene sequences. Sequences amplified from one representative isolate (PG16) have been submitted to GenBank. BLAST searches revealed that ITS (OM019317), TEF-1α (OM048103), RPB2 (ON364348), and ß-tub (OM048104) had 99 to 100% identity compared with F. graminearum (MH591453.1, KX087136.1, MF662636.1, and MZ078952.1, respectively) in GenBank. The phylogenetic analysis combined ITS - TEF-1α - RPB2 (O'Donnell et al. 2015) concatenated sequences using MEGA7.0 (Mao et al. 2021) showed the isolate was clustered with the F. graminearum clade with 100% bootstrap support (Figure 2). The isolate PG16 was used for pathogenicity tests. Ponkan fruits were surface-disinfested with 75% ethanol and rinsed with sterile distilled water three times. Then, 30 punctured wound fruits (2-mm-diameter, 2-mm-depth) with a sterile needle and 30 unwounded fruits were inoculated with conidial suspension (10 µL, 3.0 × 105 conidia/mL). while the control fruits were inoculated with 10 µL sterile distilled water. All fruits were incubated at 25°C and 90% relative humidity. Two days later, all wounded fruits inoculated with conidial suspension showed disease spots, similar symptoms to the original rotten fruits (Figure 1D). Control and conidial-inoculated unwounded fruits were healthy (Figure 1B-C). The Pathogenicity test was repeated twice, and similar symptoms were observed. Morphologically and molecularly, the re-isolated fungus matched the inoculated isolate. First report of F. graminearum causing Ponkan fruit rot in China. As Ponkan is an important citrus crop with high economic value in China, identification of the causing agent, F. graminearum, for fruit rot allows the development of control measures to manage this disease.

First report of Fusarium tricinctum causing fruit rot on navel orange (Citrus sinensis (L.) Osbeck) in China.

Citrus sinensis (L.) Osbeck is popular with consumers for its delicious taste. In December 2020, a rot symptom causing about 15% losses of a total of 450 fruits was observed on 'Newhall' navel oranges after 70 d storage (20℃, 85%-90% RH) at Jiangxi Key Laboratory for Postharvest Technology and Nondestructive Testing of Fruits & Vegetables (28.68° N, 115.85° E). The fruits were harvested from an orchard in Ganzhou City, Jiangxi Province, China (25.53° N, 114.79° E). Incipiently, the pedicles of infected fruits were brown, the peels became softened and showed yellowish-brown lesions which, gradually expanded and had white hyphae (Fig. S1A). To isolate the pathogen, the surface of diseased fruits was disinfected with NaClO (2%) for 2 min and ethanol (75%) for 0.5 min, then washed with sterile water three times. Tissues (5 × 5 mm) around the lesion were incubated on potato dextrose agar (PDA) at 28 ± 1℃ (L: D=12: 12) for 5 days. Five cultures with similar morphology were obtained and colonies initially produced white aerial hyphae and became khaki then turned pink on PDA (Fig. S1F, G, H). Abundant microconidia, macroconidia and rare chlamydospores were observed after 10 days on PDA and no glucose PDA media (Zhang et al. 2020). Macroconidia were falciform and curved to lunate, 2-4 septa, 29.38 × 3.75 µm in size (n=50) (Fig. S1K, Fig. S3). Microconidia were oval, napiform or pyriform, 0-1 septa, 12.00 × 3.43 µm in size (n=50) (Fig. S1L1 to L4, Fig. S3). Chlamydospores were found in hyphae, ellipsoidal or orbicular (Fig. S1I-1 to I-2, J-1 to J-2). The morphological features of five isolates were similar to Fusarium (Leslie and Summerell 2006). Genomic DNA of five isolates was extracted with DNA Extraction Kit (Yeasen, Shanghai, China), ITS1/ITS4, EF1Ha/EF2Tb and fRPB2-5F/fRPB2-7cR primers were used to amplify the internal transcribed spacer region (ITS), and the transcriptional elongation factor-1 alpha (TEF-1α), and RNA polymerase II (RPB2) gene sequences (White et al. 1990; Carbone and Kohn 1999; Liu et al. 1999). The ITS, TEF-1α and RPB2 sequences of five isolates were deposited in GenBank and showed 99-100% identity with corresponding sequences from F. tricinctum (Table S1). A phylogenetic tree was constructed with ITS-TEF-1α-RPB2 concatenated sequences in MEGA7.0 (Li et al. 2021) and all five isolates were placed in F. tricinctum clade with 100% bootstrap support (Fig. S2). To confirm pathogenicity, ten healthy C. sinensis fruits were surface-sterilized with 75% ethanol and inoculated with 10 µL spore suspension (1.0 × 106 spore/mL) including five wounded (with sterilized needle) and five unwounded (Fig. S1B to E). Control fruits were inoculated with 10 µL sterile water. All fruits were incubated at 28 ± 1℃, 90% RH for 7 days. The experiment was conducted three times. The lesion diameter of inoculated wounded fruits was 21.01 ± 2.52 mm and showed similar symptoms to original rotten fruits. However, the control and unwounded fruits remained healthy. To fulfill Koch's postulates, F. tricinctum was re-isolated from the inoculated fruits and deposited in Collaborative Innovation Center of Postharvest Key Technology and Quality Safety of Fruits and Vegetables in Jiangxi Province. To our knowledge, F. tricinctum has been reported on apple tree and kiwi plant in China (Zhang et al. 2021; Ma et al. 2022), but this is the first report of F. tricinctum causing fruit rot on navel orange in China. This finding provides important information for preventing postharvest disease of citrus.

First Report of Colletotrichum karstii Causing Anthracnose of Begonia lanternaria Irmsch. in China.

Begonia lanternaria Irmsch., an ornamental plant endemic in China, which is commonly used in landscape and interior decoration. In March 2021, an estimated 30% B. lanternaria plants were observed with anthracnose-like symptoms at a botanical garden conservation greenhouse in Mengla County of Yunnan Province (21.91° N, 101.21°E). Initially, small black spots developed on the disease leaves, which gradually expanded into irregular necrotic lesions surrounded by a yellowish halo, eventually turned wilting and defoliating. Twenty diseased leaves were collected and surface-disinfested with 75% ethanol for 30 s. Small fragments (5 × 5 mm) from the margin of lesions were disinfected with 1% NaClO for 120 s, washed with sterile water three times, and cultured on potato dextrose agar (PDA) at 28 ± 1℃. After 3 days single spores from four fungal colonies with identical morphology were isolated. Colonies on PDA were 70-75 mm diam in 7 d (7.5-10.6 mm/d), with dense white to gray-white mycelia attached with brown to black-brown acervulus. The underside of the culture was yellow to yellowish-brown concentric circle. Conidia were single-celled, hyaline, straight to slightly curved, cylindrical, 12.88 to 16.66 × 6.25 to 7.97 µm (av=14.65 µm × 7.22 µm, n=50) in size. For molecular identification, genomic DNA was extracted from a representative isolate, and the internal transcribed spacer, glyceraldehyde-3-phosphate dehydrogenase, calmodulin gene, ß-tublin, actin, and chitin synthase 1 genes were amplified with ITS1/ITS4 (Gardes et al, 1993), GDF/GDR (Templeton et al, 1992), CL1C/CL2C (Li et al, 2018), Bt2a/Bt2b (Prihastuti et al, 2009), ACT-512F/ACT-783R and CHS-79F/CHS-345R (Carbone et al, 1999) primers, respectively. The obtained DNA sequences showed over 99% homology with Colletotrichum karsti (GenBank Accession No. ITS: NR144790; GAPDH: KX578772; CAL: KY039988; TUB2: KX578804; ACT: LC412408; CHS1: KU251855), and the results of sequences were deposited into GenBank with accession No. MZ496954 (522/522 bp), MZ504978 (238/238 bp), MZ504979 (737/737 bp), MZ504982 (472/472 bp), MZ504981 (273/273 bp), MZ504980 (282/284 bp). The phylogenetic tree combined with ITS-ACT-GAPDH-CHS 1-CAL-TUB2 concatenated sequences using the maximum likelihood methods showed that the isolate was C. karsti. To confirm pathogenicity, Koch's postulates were conducted on intact plants, 10 µl spore suspension (1.0 × 106 conidia/ml) of each of four isolates (7-day-old culture on PDA) was inoculated on 15 wounded with a sterilized needle or non-wounded healthy living leaves, and 15 wounded leaves were inoculated with sterile water as controls. All leaves were incubated at 28 ± 1°C and 90% relative humidity (12 h/12 h light/dark). After 5 days, all wounded leaves inoculated with C. karsti showed symptoms similar to those previously observed, while the control and non-wounded leaves remained healthy. Colletotrichum karsti was re-isolated from inoculated leaves. C. karsti was previously reported to cause disease on Nicotiana tabacum L. (Zhao et al, 2020), Stylosanthes guianensis (Jia et al, 2017) and Fatsia japonica (Xu et al, 2020) in China. To our knowledge, this is the first report of C. karsti causing anthracnose of B. lanternaria Irmsch. in China. This disease reduces the ornamental and economic value of B. lanternaria Irmsch., and this work will provide a basis for the prevention and treatment of the disease in the future.

First Report of Postharvest Fruit Rot in Solanum muricatum Aiton Caused by Alternaria alternata in southwest China.

Solanum muricatum is native to South America and well known for its sweet, attractive, nutritious fruits. S. muricatum has been cultivated in China since the 1980s and increasingly popular (Li et al. 2015). In November 2021, an unknown fruit rot was observed in Shilin County of Yunnan Province (24.77 °N, 103.28 °E). The incidence of this disease was about 16% of 500 postharvest S. muricatum fruits after 7 d in storage room (25°C, 90% relative humidity). The initial symptoms were small brown spots on the fruit surface, which gradually expanded into irregular brown or black lesions, and gray-white mold developed in the center of the lesions, eventually the fruit turned rot. To isolate the pathogen, ten fruits with typical symptoms were collected and surface-sterilized with 75% ethanol for 45 s. Small fragments (5 × 5 mm) from the margin of lesions on fruit were disinfected with 1% sodium hypochlorite for 60 s, washed three times with sterile water then transferred to potato dextrose agar (PDA), and incubated at 28 ± 1℃ for 3 days (Li et al. 2022). Two fungal isolates with the same morphology were obtained and purified by single-spore isolation method. The colony was covered with thick fluffy aerial mycelia and the center was dark brown or black with white margins. Conidia were brown, pyriform or ellipsoid, with 1 to 3 longitudinal and 2 to 6 transverse septa, 15.12 to 34.01 × 6.90 to 12.73 µm (21.22 × 9.69 µm on average, n=50) in size. These morphological characteristics were consistent with Alternaria alternata (Li et al. 2015; Xiang et al. 2021; Alberto. 1992). For molecular identification, genomic DNA was extracted from a representative isolate, and primers ITS1/ITS4 (Gardes et al. 1993), TEF-F/TEF-R (Lawrence et al. 2013), Alt-F/Alt-R (Hong et al. 2005), GPD-F/GPD-R (Berbee et al. 1999) and EPG-F/EPG-R (Peever et al. 2004) were used to amplify the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF), Alternaria major allergen (Alt a1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and endo-polygalacturonase (endoPG), respectively. The obtained DNA sequences (ITS: OM049821; TEF: OM069656; Alt a1: OM069655; GAPDH: OM069654 and endoPG: OM069653) showed over 99% homology with that of A. alternata (GenBank Accession No. MN856355.1 (565/573 bp); MN258023.1 (267/267 bp); KY923227.1 (491/501 bp); LC131645.1 (608/609 bp) and MN698284.1 (452/454 bp)). A phylogenetic tree based on the combined ITS, TEF, Alt a1, GAPDH, and endoPG sequences using the maximum likelihood methods with Kimura 2-parameter model, bootstrap nodal support for 1000 replicates in MEGA7.0 (Li et al. 2019) revealed that the isolate was assigned to A. alternata. To confirm pathogenicity, 10 µL spore suspension (1.0 × 106 conidia/ml) obtained from 7-day-old PDA cultures of each isolate were inoculated on 15 needle-wounded and 15 non-wounded surface-disinfected fruits, respectively. Healthy fruits were inoculated with sterile water as controls and the experiment was repeated 3 times. All fruit were incubated at 25 ± 1℃, 90% relative humidity. After 7 days, all the wounded and non-wounded fruit inoculated with A. alternata showed similar symptoms to those observed on the previously fruits, while the control fruits remained healthy. The same pathogen was again isolated from the inoculated fruits, thus Koch's postulates were fulfilled. A. alternata causing fruit rot of Prunus avium and Mangifera indica in China were reported in previous studies (Ahmad et al. 2020; Liu et al. 2019). As far as we know, this is the first report of postharvest fruit rot on S. muricatum caused by A. alternata in southwest China. This work provides a basis for the development of control strategies of the disease in the future.

First Report of Alternaria alternata causing fruit Rot on Tetradium ruticarpum in China.

Tetradium ruticarpum, previously and commonly known as Evodia rutaecarpa, is a tree that produces a fruit which is one of the most important traditional Chinese medicine herbs in China (Zhao et al. 2015). In July 2019, an investigation of diseases of T. ruticarpum was conducted in the farmland of Ruichang County (29.68° N, 115.65° E), Jiujiang City, China. An unknown fruit rot disease was observed and the incidence rate was estimated to be 60% to 70% within a 5,000 m2 area. The early symptoms appeared as small circular to irregular dark brown or black spots on the fruit, which gradually coalesced to a light brown-to-black discoloration and caused fruit rot. To identify the causal agent of the disease, 10 diseased fruits were collected and surface disinfected with 2% sodium hypochlorite for 2 min, 70% ethanol for 30 s, rinsed in sterile water and dried on filter paper. Tissues from non-symptomatic tissue as well as from the margin between healthy and affected edge were incubated on potato dextrose agar (PDA) at 25±1°C (12 h light/dark) with 90% relative humidity for 5 days. The colonies were brown to black with abundant whitish margins. Conidiophores were brown and measured 20.40 - 43.10×1.30 - 4.20 µm (25.47 × 2.35 µm on average, n=50). Conidia produced in single or branched chains, were obclavate or ovoid, approximately 9.90 - 32.80×6.50 - 14.50 µm (28.75×12.57 µm on average, n=50) with 2 to 5 transverse septa and 0 to 3 longitudinal septa. The colonies were consistent with Alternaria alternata (Simmons 2007). For molecular identification, the f partial internal transcribed spacer (ITS) regions, Glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes, translation elongation factor 1-alpha (TEF) and Alternaria major allergen (Alt a1) gene of the isolate were amplified using primers ITS1/ITS4 (White et al. 1990), GDF/GDR (Templeton et al. 1992), EF1-728F/EF1-986R (Carbone and Kohn 1999) and Alt-for/Alt-rev (Hong et al. 2005). Sequence data showed 100% homology to A. alternata (GenBank accessions No.MN625176.1 (570/570 bp), MK683866.1 (618/618 bp), MK637432.1 (281/281 bp), KT315515.1 (488/488 bp)), respectively and the sequence data were deposited into GenBank with accession numbers MN897753 (ITS), MT041998 (gapdh), MT041999 (TEF), and MT042000 (Alt a1). Based on both morphological and molecular characteristics, the pathogen was identified as A. alternata. To confirm pathogenicity, 10 µl of a spore suspension (1.0 × 106 conidia/ml) obtained from 5-day-old PDA cultures of the strain were inoculated on 20 wounded (using sterile needle) and 20 nonwounded healthy T. ruticarpum fruits previously disinfected in 75% ethanol. Control fruits including 20 wounded fruits and 20 nonwounded fruits were inoculated with sterilized water. All fruits were incubated at 25±1°C (12 h light/dark) with 90% relative humidity. Four days later, all the wounded and non-wounded fruits showed the initial symptoms of black rot which was similar to that observed in the field, while the wounded and nonwounded fruits treated with sterile water remained healthy. The same pathogen was again isolated from the inoculated fruits. The pathogenicity experiment was repeated three times with the same results. As far as we know, this is the first report of A. alternata causing fruits rot on T. ruticarpum in China, and the identification of the pathogen will provide useful information for developing effective control strategies.