First Report of Black Scurf Caused by Rhizoctonia solani AG 2-2IV on Potato tubers in Heilongjiang Province, China.

Black scurf and stem canker on potato (Solanum tuberosum L.), caused by Rhizoctonia solani, is one of the most important soil-borne diseases throughout the world. Isolates of R. solani anastomosis group (AG) 3-PT have been reported as the predominant cause of the disease on potato (Carling 1996) and the same results were also obtained in Heilongjiang Province, China (Yang et al. 2017). In October 2020, 14 diseased potato tubers (cv. Youjin-885) with symptoms typically associated with black scurf were found in Hegang City of Heilongjiang in Northeast China, where potatoes are grown for propagation in the breeding nursery. Pieces of sclerotia were removed from the surface of the potato and were surface sterilized with 70% ethanol for 30 s and 0.5% NaClO for 1 min, then rinsed three times with sterile distilled water and placed on potato dextrose agar (PDA) at 25°C in the dark. After incubation for 48 to 72 h, mycelia resembling Rhizoctonia were microscopically examined for morphological characteristics, and hyphal tips transferred to fresh plates of PDA. The characteristics of the observed isolate were typical of R. solani Kühn, which include hyphal branching at right angles, a septum near the branching point and a slight constriction at the branch base (Yang et al. 2015). Hyphal cells were also determined to be multinucleate by staining with 1% safranin O and 3% KOH solution (Bandoni 1979). PCR amplification and DNA sequencing of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) was performed by using the universal primers ITS4/ITS5 (White et al. 1990). The resulting sequence of 700 bp (GenBank accession no. OL770460) showed more than 99% identity to AG 2-2IV isolates present in GenBank (e.g. AB911322; KR259910). On the basis of morphological characteristics and molecular analysis, the isolate was identified as R. solani AG 2-2IV. Pathogenicity of the isolate was tested in greenhouse conditions. Pathogen-free minitubers (cv. Favorita) of approximately the same size (10 to 20 g) were allowed to sprout at room temperature for 10 days. The minitubers were then planted in autoclaved soil in a plastic pot (4 L capacity), placed in a greenhouse at 18 - 27°C (night-day) with 50% relative humidity and watered as required. The pots were inoculated with 7-mm-diameter mycelial plugs (from one PDA petri plate) near the minituber, which was then covered with potting mix. The control pots were inoculated with sterile plugs of PDA. Each treatment consisted of 10 plants, and the experiment was repeated three times. Two months after stems emerged, plants and progeny tubers were harvested and assessed for disease. Stem cankers typical of R. solani infection and black scurf were observed on plants grown in pots inoculated the mycelial plugs, but the control plants remained disease free. Fungi reisolated from symptomatic stems and tubers were identified as R. solani AG 2-2IV using morphological characters and ITS sequences.Sclerotia were observed on PDA by incubating at 25oC in the dark. Although eight AGs have been previously shown to cause black scurf and stem canker in Heilongjiang (Li et al. 2014; Yang et al. 2015; Yang et al. 2017; Yang et al. 2019; Yang et al. 2020), to our knowledge, this is the first report of AG 2-2IV causing disease on potatoes in Heilongjiang Province, the main potato seed production area of China. Early detection of R. solani AG 2-2IV during potato seed production is necessary to prevent its dispersal via infected tubers to other fields across China. The information of which AG is present will assist in developing management strategies for this disease.

Streptomyces rhizophilus Causes Potato Common Scab Disease.

Common scab (CS) caused by Streptomyces spp. is a significant soilborne potato disease that results in tremendous economic losses globally. Identification of CS-associated species of the genus Streptomyces can enhance understanding of the genetic variation of these bacterial species and is necessary for the control of this epidemic disease. The present study isolated Streptomyces strain 6-2-1(1) from scabby potatoes in Keshan County, Heilongjiang Province, China. PCR analysis confirmed that the strain harbored the characteristic Streptomyces pathogenicity island (PAI) genes (txtA, txtAB, nec1, and tomA). Pathogenicity assays proved that the strain caused typical scab lesions on potato tuber surfaces and necrosis on radish seedlings and potato slices. Subsequently, the strain was systemically characterized at morphological, physiological, biochemical, and phylogenetic levels. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 6-2-1(1) shared 99.86% sequence similarity with Streptomyces rhizophilus JR-41T, isolated initially from bamboo in rhizospheric soil in Korea. PCR amplification followed by Sanger sequencing of the 16S rRNA gene of 164 scabby potato samples collected in Heilongjiang Province from 2019 to 2020 demonstrated that approximately 2% of the tested samples were infected with S. rhizophilus. Taken together, these results demonstrate that S. rhizophilus is capable of causing potato CS disease and may pose a potential challenge to potato production in Heilongjiang Province of China.

Potato calcineurin B-like protein CBL4, interacting with calcineurin B-like protein-interacting protein kinase CIPK2, positively regulates plant resistance to stem canker caused by Rhizoctonia solani.

Introduction: Calcium sensor calcineurin B-like proteins (CBLs) and their interacting partners, CBL-interacting protein kinases (CIPKs), have emerged as a complex network in response to abiotic and biotic stress perception. However, little is known about how CBL-CIPK complexes function in potatoes. Methods: In this study, we identified the components of one potato signaling complex, StCBL4-StCIPK2, and characterized its function in defense against Rhizoctonia solani causing stem canker in potato. Results: Expressions of both StCBL4 and StCIPK2 from potato were coordinately induced upon R. solani infection and following exposure to the defense genes. Furthermore, transient overexpression of StCBL4 and StCIPK2 individually and synergistically increased the tolerance of potato plants to R. solani in Nicotiana benthamiana. Additionally, the transgenic potato has also been shown to enhance resistance significantly. In contrast, susceptibility to R. solani was exhibited in N. benthamiana following virus-induced gene silencing of NbCBL and NbCIPK2. Evidence revealed that StCBL4 could interact in yeast and in planta with StCIPK2. StCBL4 and StCIPK2 transcription was induced upon R. solani infection and this expression in response to the pathogen was enhanced in StCBL4- and StCIPK2-transgenic potato. Moreover, accumulated expression of pathogenesis-related (PR) genes and reactive oxygen species (ROS) was significantly upregulated and enhanced in both StCBL4- and StCIPK2- transgenic potato. Discussion: Accordingly, StCBL4 and StCIPK2 were involved in regulating the immune response to defend the potato plant against R. solani. Together, our data demonstrate that StCBL4 functions in concert with StCIPK2, as positive regulators of immunity, contributing to combating stem canker disease in potato.