First Report of Root and Bulb Rot Caused by Rhizoctonia solani AG-6 on Lanzhou Lily (Lilium davidii var. unicolor) in China.

Lanzhou lily (Lilium davidii var. unicolor) is the only famous sweet lily variety that has high edible, medicinal and ornamental value in China, which is mostly planted in the middle areas of Gansu Province in China. In recent years, severe yellowing and wilting of leaves, stem wilt, root and bulb rot symptoms were observed on Lanzhou lily in Qilihe District, Lanzhou, which has resulted in serious loss of bulb production. From June to August 2022, a survey of Lanzhou lily disease was carried out in Xiguoyuan and Weiling township of Qilihe District, Lanzhou. Typical symptoms of root and bulb rot were observed in Lanzhou lily fields. The disease incidence was estimated up to 30%. Fragments of symptomatic roots and bulbs were surface sterilized with 75% ethanol for 10 s, 2% sodium hypochlorite for 2 min, washed three times with sterilized distilled water, and then blotted dry on sterile filter paper. Fragments were placed on PDA medium and incubated at 25 ± 1°C in darkness for 5 days and 2 isolates were purified by the single-tip culture. Colonies of the fungus were white initially, and then turned light brown to brown, raised, and with entire or undulate edges. Sclerotia were brown and produced on PDA after 25 days of incubation at 25 ± 1°C in the dark. Genomic DNA from each of the two isolates was extracted, and the internal transcribed spacer (ITS) region was amplified and sequenced with the primer pair ITS5/ITS4 (White et al. 1990). The sequences of strains QLH22LD01 and QLH22LD02 were deposited in GenBank (OR710804 and OR710805). Phylogenetic analyses were performed using the Maximum Likelihood method with ITS sequences for anastomosis groups (AG) of Rhizoctonia solani. The phylogenetic tree grouped the two isolates within the R. solani AG-6 clade with high bootstrap support (100%). PCR analysis was performed with 21 primers specifically designed to detect individual anastomosis groups or anastomosis subgroups of R. solani (Carling et al., 2002; Misawa and Kurose, 2019; Misawa et al., 2020; Okubara et al., 2008). Among the 21 specific primer pairs, only AG-6 specific primer amplified the fungal DNA, indicating that the two isolates tested belonged to the R. solani AG-6. Therefore, these two strains were identified as R. solani AG-6. For pathogenicity tests, two isolates were grown individually on sterile wheat kernels at 25 ± 1°C for 14 days. Certified pathogen-free Lanzhou lily bulbs were grown in the plastic pot filled with the sterilized soil. Fifteen 2- week-old plants were inoculated by digging the soil and burying ten infested wheat kernels in the soil adjacent to the roots. Control plants were inoculated with sterile wheat kernels using the same procedure. All plants were placed in a greenhouse with a 12h/12h light/dark photoperiod at 15 to 30°C. Fifty days after inoculation, typical root and bulb rot symptoms developed on all inoculated plants, similar to symptoms observed in the field, whereas control plants remained symptomless. Pathogenicity test was performed three times with similar to symptoms observed in the field. Finally, the fungi were reisolated from the symptomatic plants and identified by molecular analysis as the isolates used for inoculation, thus fulfilling Koch's postulates. To our knowledge, this is the first confirmed report of R. solani AG-6 causing root and bulb rot on Lanzhou lily in China. Our findings improve knowledge about R. solani AGs occurring in Lanzhou lily fields in China. Due to serious damages caused by this disease in recent years in China, further studies should be conducted to investigate the diversity, prevalence, disease control measures and fungicide sensitivity of AGs distributed in the main Lanzhou lily-producing states in China.

First Report of Neck Rot Caused by Botrytis aclada on Onion (Allium cepa L.) in Gansu Province, China.

Onion (Allium cepa L.) is one of the most important cultivated vegetable crops in many countries, including China (Hanci 2018; Steentjes et al. 2021). Gansu, in the northwest region of China, is a major area of onion production (Zhang et al. 2022). In October 2021, typical symptoms of neck rot were observed on stored onion bulbs (cv. Honghe) in Jiuquan, Gansu Province, China. Further surveys of 20 bags of onion bulbs randomly selected in a storage facility with bulbs harvested from 73 ha indicated that approximately 5% of the bulbs had typical neck rot symptoms. The neck of infected bulbs developed a water-soaked decay, with softened and discolored inner scales with white to gray mycelium in the neck. Infection usually began in the neck and sometimes spread through the entire bulb. In severely affected bulbs, the fleshy scales were span style="font-family:'Times New Roman'">decayed and the bulbs had shrunk, with black sclerotia between the rotting scales in the neck and shoulders of the bulb. Small pieces cut from the margins of lesions were surface-disinfested with 75% ethanol for 10 s, and 1% NaClO for 1 min, rinsed three times in sterile distilled water, dried on sterile filter paper, placed onto potato dextrose agar (PDA), and incubated at 20 ± 1℃ for 5 days in the dark. Ten pure cultures were obtained by single-spore isolation (one from each of 10 bulbs). All 10 isolates produced colonies that initially were white, and became gray to dark in color with gray mycelium that was covered with abundant conidia resembling Botrytis species when cultured on PDA at 20 ± 1℃ for 10 days in the dark. Conidia (n = 100 per isolate) were one-celled, ellipsoid or ovoid, beige to dark brown, and 5.6 to 14.8 × 3.9 to 8.5 µm. Sclerotia were not produced by any of the isolates on PDA after incubation at 20 ± 1°C for 30 days. The internal transcribed spacer (ITS) region of ribosomal DNA, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene, heat shock protein 60 (HSP60) gene, and DNA-dependent RNA polymerase II core subunit (RPB2) gene of two representative isolates, JQ21AC03 and JQ21AC09, were amplified and sequenced with primers ITS1/ITS4, G3PDH-F/G3PDH-R, HSP60-F/HSP60-R, and RPB2-F/RPB2-R, respectively (Staats et al. 2005), and deposited in GenBank (ITS: OP604277 and OP604283; G3PDH: OP627512 and OP627515; HSP60: OP627513 and OP627516; and RPB2: OP627514 and OP627517). BLASTn analysis of the resulting sequences showed 98 to 100% similarity with those of Botrytis aclada. A maximum likelihood phylogenetic tree generated by combining the sequenced loci using MEGA11.0, clustered isolates JQ21AC03 and JQ21AC09 with B. aclada with 99% bootstrap support. Based on these results, isolates JQ21AC03 and JQ21AC09 were identified as B. aclada. Pathogenicity of the two isolates on the onion cv. Honghe was confirmed by inoculating 30 heathy onion bulbs per isolate with 100 µL of conidial suspension (1×105 conidia/ml), while control bulbs were inoculated with 100 µL sterile distilled water. All 30 bulbs inoculated with each isolates developed neck rot symptoms after 30 days incubation at 15 ± 1ºC in the dark, with symptoms identical to those observed in the original storage facility, whereas control bulbs remained symptomless. The pathogen was re-isolated from the symptomatic tissue of inoculated bulbs, fulfilling Koch's postulates. hang et al (2008) documented this pathogen causing bulb rot of onions in Wuhan, Hubei Province, 1,700 km from Gansu. To the best of our knowledge, this is the first report of B. aclada causing neck rot on onion bulbs in storage in Gansu Province, which is the main onion production region in China. Considering that onion is the main source of income for growers in Gansu, further studies will be required to understand the epidemiology of this disease and foster appropriate disease management measures to avert disease outbreaks in the future.

First Report of Anthracnose Disease Caused by Colletotrichum fioriniae on Barbary wolfberry (Lycium barbarum L.) in Gansu Province, China.

Barbary wolfberry (Lycium barbarum L.) is a well-known edible and medicinal plant, widely grown in northwest China (Gao et al. 2021). During the summer of 2019, typical anthracnose symptoms were observed on fruits of barbary wolfberry in Baiyin, Gansu province, China. Approximately 30% of the barbary wolfberry fruits had typical anthracnose symptoms. Lesions on barbary wolfberry fruits were dark, circular or irregular, sunken, and necrotic or wilted, with the presence of orange to pink conidial masses under high humidity. Small pieces cut from the margins of lesions were surface disinfested with 75% ethanol for 10 s, and 1% NaClO solution for 1 min, rinsed three times in sterile distilled water, dried on sterile filter paper, placed onto potato dextrose agar (PDA), and incubated at 25 ± 1℃ for 5 days in the dark. The pure cultures were obtained by single-spore isolation. All isolates produced pale gray and dense aerial mycelia, in reverse orange to red, at times showing concentric rings on PDA at 25℃ after 10 days in the dark. Conidia (n=100) were colorless, smooth-walled, aseptate, fusiform elliptical with one or both ends, and 8.3 to 17.6 × 3.7 to 6.2 µm. Appressoria (n = 100) were solitary, pale to medium brown, smooth-walled, subglobose to elliptical, sometimes clavate or irregular, and 5.7 to 11.7 × 4.1 to 8.5 µm. The internal transcribed spacer (ITS) region of ribosomal DNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, bate-tubulin 2 (TUB2) gene, actin (ACT) gene, calmodulin (CAL) gene, chitin synthase 1 (CHS-1) gene, and histone H3 (HIS3) gene of the two representative isolates BY19LB02 and BY19LB06 were amplified and sequenced with primers ITS1/ITS4, GDF1/GDR1, T1/Bt2b, ACT-512F/ACT-783R, CL1/CL2A, CHS-79F/CHS-354R, CYLH3F/CYLH3R, respectively (Damm et al. 2012), and deposited on GenBank (ITS, MZ496816 and MZ505524; ACT, MZ557422 and MZ557417; CHS-1, MZ557423 and MZ557418; GAPDH, MZ557424 and MZ557419; HIS3, MZ557425 and MZ557420; TUB2, MZ557426 and MZ557421). BLAST analysis of the resulting for all the sequences showed 98 to 100% similarity with those of C. fioriniae. Based on the above, the isolates BY19LB02 and BY19LB06 were identified as C. fioriniae. To confirm the pathogenicity, detached heathy barbary wolfberry fruits were surface-sterilized with 75% ethanol for 30s, rinsed three times in sterile distilled water, allowed to dry on sterile filter paper, and then wounded using sterilized needles. Fruits were inoculated by pipetting 10 µL of conidial suspension (1×106 conidia/mL) onto each wound, and controls were inoculated with 10 µL sterile distilled water. Each treatment had 30 fruit replicates. These fruits were kept in a moist chamber at 28°C in the dark. The experiment was repeated three times. After 5 days, anthracnose symptoms were observed on all of the inoculated fruits and identical to those observed in the field, whereas control fruits did not develop symptoms. Theathogen was re-isolated from the lesions of inoculated fruits, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of C. fioriniae causing anthracnose on barbary wolfberry in Gansu Province, China. The same disease on barbary wolfberry was reported in Jilin Province, China (Liu et al. 2016). Gansu is one of the main barbary wolfberry producing areas in northwest China and its geographical area, climate and environmental conditions are different from Jilin Province. Considering that barbary wolfberry is the main source of income for growers in Gansu, this identification can aid in the selection of appropriate management measures for this disease.