First Report of Leaf Spot Caused by Alternaria alternata on Chard (Beta vulgaris var. cicla L.) in China.

Chard (Beta vulgaris var. cicla L.) is popular vegetable in China. In June 2023, a leaf spot disease was observed on Chard plants in Hunan Province (27°46'10.99″N, 112°05'52.80″E), China. The disease incidence was 30% in a surveyed of about 500 plants. Symptoms began as many light brown round- to polygon-shaped spots on chard leaves, then developed and enlarged into grayish-white lesions, with the edge of the spots brown to dark brown. A total of 10 symptomatic samples were randomly collected. To identify the pathogen, symptomatic tissues (0.5 × 0.5 cm) from the lesion margin surface were sterilized with 75% ethanol for 30 s and 2% NaClO for 1 min, rinsed 3 times with sterile water, air dried. The sterile pieces were placed on potato dextrose agar (PDA) and incubated at 25°C. A total of nine isolates were obtained. Fungal colonies cultured on potato carrot agar (PCA) were almost the same as each other, and two representative isolates (TC0, TC10) were used for further identification. On PCA, the fungal hyphae were initially white and finally gray-brown with flocculent aerial mycelia. Conidia were solitary or in chains, with various shapes, mostly subglobose, the size was 13.2 to 28.0 µm long and 5.8 to 13.0 µm wide (n = 30). The cultural and morphological characteristics of isolates were similar to those of Alternaria sp (Simmons et al. 2007). For molecular identification, four loci, ITS (White et al. 1990), RPB2 (O'Donnell, 2022), H3 (Zheng et al. 2015), and GAPDH (Berbee et al. 1999), were sequenced from two representative isolates (TC0, TC10). Compared with a reference isolate, Alternaria alternata strain CBS 107.27, GenBank accession nos. KP124300.1 (ITS), KP124768.1 (RPB2), KP124157.1 (GAPDH). The ITS, RPB2, and GAPDH sequences of TC0 and TC10 showed 99% (502 of 504 bp ), 100% (753 of 753 bp), and 99% (560 of 561 bp) similarity, respectively. Compared with a reference isolate, A. alternata isolate 21-5, GenBank accession no. MN840996.1 (H3), H3 sequences of TC0 and TC10 showed 99% (399 of 401 bp) similarity. The sequences of two isolates (TC0, TC10) were deposited in GenBank with accession numbers PP837733.1, PP565404.1(ITS), PP839298.1, PP573905.1(RPB2), PP839299.1, PP573904.1 (GAPDH), and PP839297.1, PP573903.1(H3). Phylogenetic trees were constructed using the sequences and showed that isolates (TC0, TC10) were in the same clade with A. alternata strains. TC0 and TC10 were identified as A. alternata based on the morphological characteristics and molecular phylogeny. Pathogenicity testing was conducted on six-month-old healthy plants, (cv. Green Stalk), three plants were inoculated by spraying spore solution (1 × 106 conidia/mL), and three plants were sprayed with sterile water as a control. The pathogenicity test was performed 3 times. Plants were maintained at 28°C and >80% RH. Plants showed symptoms after 30 days, symptoms were observed similar to those of the original infected plants, control plants were asymptomatic. The fungus was reisolated, confirmed as A. alternata based on conidial characteristics, no pathogenic fungus was isolated from the control plants. A. alternata has previously been reported on beet (also Beta vulgaris) in China (Tai, F. L. 1979; Zhuang, W. Y. 2005). To our knowledge, this is the first report of leaf spot caused by A. alternata on chard in China. This result may expand the etiological study of A. alternata and the control strategy of Chard leaf spot.

First report of Nigrospora musae causing white leaf spot of Basella alba L in China.

Basella alba L, an edible annual twining herb of the genus Basella and the family Basella, has been widely introduced and cultivated in China. Basella alba L. as a leaf vegetable, is rich in vitamins A and C, iron, and calcium (FAO 1988). In May 2022, severe white leaf spots were observed in plantation located in Shuangfeng County (27°41'36" N, 111°56'60" E), Hunan Province, China. More than 50 Basella alba L plants were surveyed with over 80% disease incidence in an area of 300 square meters of greenhouse. The symptoms on leaves were initially small purplish-brown lesions from leaf margins or tips, with lesions expanded, the middle of the lesions was yellowish-white to yellowish-brown, slightly dented. The edge of lesions was purplish-brown, with obvious boundary between the diseased parts and the non-diseased ones. A total of 20 symptomatic samples were randomly collected. Lesion margins were surface sterilized in 2% sodium hypochlorite for 1 min, rinsed with sterile distilled water for three times, dried, placed on potato dextrose agar (PDA), and incubated at 25°C and 60% relative humidity in the dark for 3 days. Hyphal sections from colony edges were transferred to new PDA plates. Six isolates were obtained. Colonies were fast-growing, massive sparse aerial hyphae, initially white, turning gray and black after 7 days. Hyphae were branched, septa, and transparent. To induce sporulation, colonies were transferred to sodium carboxymethyl cellulose (CMC) plates (Z. M. Wen., & X. Y. Luo 1991). Conidia were single-celled, dark black, oblate, or nearly spherical, and measured 10.2 to 15.1 µm × 9.7 to 16.0 µm in diameter (n=50). For molecular identification, the rDNA internal transcribed spacer (ITS), the ß-tubulin gene (TUB), and the translation elongation factor 1-alpha gene (TEF1) were amplified from genomic DNA by primers ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass & Donaldson. 1995), and EF1-728F/EF1-986R (Carbone & Kohn, 1999). The sequences of six isolates (L1, L2, L7, L10, L11, L12) were deposited in GenBank with accession numbers OP703335, OP703336, OP703337, OP703338, OP703339, OP703340 (ITS), OP784252, OP784157, OP784253, OP784254, OP784255, OP784256 (TEF-1α), and OP724156, OP724158, OP779771, OP779772, OP779773, OP779774 (TUB2). A blast search of sequences showed the ITS, TEF-1α, and TUB2 sequences had >98% identity with homologue sequences from Nigrospora musae isolates BRJ2 (OP451019.1), CBS 319.34 (KY019419.1) and LC6385 (KY019567.1), respectively. These morphological features and molecular identification indicated that the pathogen possessed identical characteristics as Nigrospora musae (Wang, 2017). Pathogenicity test was carried out in plants. Strains were cultured on CMC plates for 14 days, then the mycelium was scraped to make conidial suspension (1×106 conidia/mL). After 5-6 leaves of the Basella alba L were sprouted, conidial suspension was sprayed directly on the leaves, with leaves sprayed by sterile distilled water as the control. All plants were kept in the greenhouse with temperature at 25/30°C (night/day) and 75% relative humidity. After 7 days, symptoms were observed on inoculated leaves of plants, which were the same as previously described samples, while the control plants showed no symptoms. The test was repeated three times with similar results. The strains reisolated from the inoculated leaves were morphologically identical to Nigrospora musae, conforming to Koch's postulates. symptoms of Nigrospora musae is similar to that of the other leaf diseases of Basella alba L reported in China. (H. P. Jiang.2000; S. Tan.1996). To our knowledge, this is the first report of Nigrospora musae causing white leaf spot of Basella alba L in China. The pathogen may severely threat the production of Basella alba L. The information on identification of this fungus may be helpful to the control and prevention of the disease. References: 1. FAO. 1988. Page 103 in: Traditional Food Plants: A Resource Book for Promoting the Exploitation and Consumption of Food Plants in Arid, Semi-arid and Sub-humid Lands of Eastern Africa. FAO Food and Nutrition Paper 42. FAO, Rome, Italy. 2. Z. M. Wen., & X. Y. Luo. Fusarium graminearum spore production medium filtering [J]. Chinese journal of food hygiene, 1991 (04): 11-13. 3. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. 4. Carbone, I., et al. 1999. Mycologia. 91: 553-556. 5. Glass, N. L., and Donaldson, G. C. 1995. Appl. Environ. Microbiol. 61: 1323. 6. Wang, 2017. Phylogenetic reassessment of Nigrospora: Ubiquitous endophytes, plant, and human pathogens. 7. H. P. Jiang., et al. Occurrence and comprehensive control of white leaf spot of Basella alba L [J]. Plant Protection Technology and Extension, 2000(02):19. 8. S. Tan. The symptoms and control measures of white leaf spot of Basella alba L [J]. Anhui Agricultural, 1996(08):15. *Indicates the corresponding author. Kaifa Guo, E-mail: andygkf@126.com.

First report of Botryosphaeria dothidea causing leaf spot of Camellia oleifera in China.

Camellia oleifera Abel., a small evergreen tree or shrub, is mainly distributed in central and southern China with a larger scale of 4.5 × 106 hectares (Zhu 2020). In May 2021, severe leaf spots were observed in plantation located in Shuangfeng County (27°41'36" N, 111°56'60" E), Hunan Province, China. More than 60 C. oleifera plants were surveyed with over 80% disease incidence. The symptoms on leaves were initially small brown lesions from leaf margins or tips, developing to suborbicular or irregular-shaped dark brown lesions, leading to leaves withered. A total of 60 symptomatic samples were randomly collected. Lesion margins were surface sterilized in 2% sodium hypochlorite for 1 min, rinsed with sterile distilled water for three times, dried, placed on potato dextrose agar (PDA), and incubated at 25°C in the dark for 3 days. Hyphal sections from colony edges were transferred to new PDA plates. Three isolates of Botryosphaeria dothidea were obtained. Colonies of B. dothidea were initially white gradually turning dark-gray with dense aerial mycelium after 6 days. To induce sporulation, colonies of YCB17 were transferred to synthetic nutrient-poor agar (SNA) with sterilized leaves of C. oleifera. Cultures were initially incubated at 25°C in the dark for 3 days, then alternatively exposed to 12-hours near-UV light and 12-hours white light (CHU et al. 2021). After 5 days, conidia formed on leaves were examined microscopically. The conidia were unicellular, aseptate, hyaline, and fusoid, 20.9-25.5×4.7-6.4 µm (n = 50). Morphological characteristics of the isolates matched the description of B. dothidea (Slippers et al. 2014). DNA sequence was amplified using primer pairs ITS1/ITS4 (Tang et al. 2022), EF1-728F/986R (Slippers et al. 2004), and ßt2a/2b (Glass & Donaldson. 1995) respectively. The sequences of three isolates (YCB2, YCB3, YCB17) were deposited in GenBank with accession numbers ON714603, MZ613350, MZ613349 (ITS), OM328342, OM328343, OM328344 (TEF-1α), and OM328345, OM328346, OM328347 (TUB2). A blast search of sequences showed the ITS, TEF-1α, and TUB2 sequences had >99% identity with homologue sequences from B. dothidea isolates IRNHM-KZ49 (MG198191.1), CAP288 (EF638732.1) and Mu1 (MK423987.1), respectively. For pathogenicity testing, healthy leaves of 2-year-old C. oleifera plants in the greenhouse were spray-inoculated with conidial suspension (2×106 conidia/mL) from YCB17. Ten surface-sterilized and wounded leaves per plant were sprayed with 30 µL suspension. The other ten wounded leaves sprayed with sterile distilled water served as control. All plants were kept in the greenhouse with temperature at 26 ± 2°C and 50% relative humidity. After 12 days, initial symptoms were observed on more than 80% leaves inoculated with conidial suspension, whereas no symptoms were observed on the control leaves. The test was repeated three times with similar results. It was found that B. dothidea could cause leaf spot of C. oleifera. The infected leaves showed same symptom as samples. Re-isolated fungi from infected leaves were morphologically identical to B. dothidea. Botryosphaeria dothidea has been reported causing leaf spot in a wide range of hosts, but has not previously been reported causing disease on C. oleifera. To our knowledge, this is the first report of B. dothidea causing leaf spot of Camellia oleifera in China. The information on identification of this fungus may be helpful to the control and prevention of the disease. References: 1. Chu Rui-Tian, et al. 2021. Mycosystema 40(3): 473. 2. Glass, N. L., and Donaldson, G. C. 1995. Appl. Environ. Microbiol. 61: 1323. 3. Slippers, B., et al. 2004. Mycologia 96:83. 4. Slippers, B., et al. 2014. Persoonia 33:155. 5. Tang, Y., et al. 2022. Plant Dis. 106: 765. 6. Zhu P.X. People's Daily. 2020.11.09. http://gz.people.com.cn/n2/2020/1119/c194844-34425098.html. *Indicates the corresponding author. Kaifa Guo, E-mail: andygkf@126.com.