Huanglongbing (HLB), also known as citrus greening, is the most destructive disease of citrus worldwide. HLB is associated with the non-culturable bacterium, Candidatus Liberibacter asiaticus (CaLas) in the United States. The virulence mechanism of CaLas is largely unknown, partly because of the lack of a mutant library. In this study, Tobacco mosaic virus (TMV) and Nicotiana benthamiana (N. benthamiana) were used for large-scale screening of the virulence factors of CaLas. Agroinfiltration of 60 putative virulence factors in N. benthamiana led to the identification of four candidates that caused severe symptoms in N. benthamiana, such as growth inhibition and cell death. CLIBASIA_05150 and CLIBASIA_04065C (C-terminal of CLIBASIA_04065) could cause cell death in the infiltrated leaves at five days post infiltration. Two low-molecular-weight candidates, CLIBASIA_00470 and CLIBASIA_04025, could inhibit plant growth. By converting start codon to stop codon or frameshifting, the four genes lost their harmful effects to N. benthamiana. It indicated that the four virulence factors functioned at the protein level rather than at the RNA level. The subcellular localization of the four candidates was determined by confocal laser scanning microscope. CLIBASIA_05150 located in the Golgi apparatus; CLIBASIA_04065 located in the mitochondrion; CLIBASIA_00470 and CLIBASIA_04025 distributed in cells as free GFP. The host proteins interacting with the four virulence factors were identified by yeast two-hybrid. The host proteins interacting with CLIBASIA_00470 and CLIBASIA_04025 were overlapping. Based on the phenotypes, the subcellular localization and the host proteins identified by yeast two-hybrid, CLIBASIA_00470 and CLIBASIA_04025, functioned redundantly. The hypothesis of CaLas virulence was proposed. CaLas affects citrus development and suppresses citrus disease resistance, comprehensively, in a complicated manner. Ubiquitin-mediated protein degradation might play a vital role in CaLas virulence. Deep characterization of the interactions between the identified virulence factors and their prey will shed light on HLB. Eventually, it will help in developing HLB-resistant citrus and save the endangered citrus industry worldwide.

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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.

IntroductionCalcium 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.MethodsIn 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.ResultsExpressions 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.DiscussionAccordingly, 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.

Rhizoctonia solani is one of the most important soil borne pathogenic fungi, causing disease in several important crops worldwide. The symptoms of R. solani infection are attributed to various plant diseases, such as damping off, root rot, stem cankers, crown rot and blights (Ogoshi 1996). In potato, R. solani has been associated with black scurf and stem cankers causing qualitative and quantitative damage to infected crops (Djébali and Belhassen 2010). Isolates of R. solani AG3 have been reported as the predominant cause of stem canker on potato (Carling 1996) and the same results were also obtained in the cities of Harbin and Suihua in Heilongjiang Province, China (Yang et al. 2017). During the summer of 2019, diseased potato plants (cv. Youjin-885) with symptoms typically associated with stem canker (Supplementary Fig. 1A) were found in Jiamusi City of Heilongjiang in Northeast China, where potatoes are grown widely. Stem pieces (5 mm long) were taken from the margins of the healthy and diseased tissues and were surface sterilized with 70% ethanol for 30 s and 0.5% NaClO for 1 min, then rinsed 3 times with sterile distilled water and placed on potato dextrose agar (PDA) at 25°C in the dark. After incubation for 48–72 h, hyphae tips resembling Rhizoctonia were microscopically examined for morphological characteristics, and 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 (Supplementary Fig. 1B), amongst other characteristics. Hyphal cells were also determined to be multinucleate by staining with 1% safranin O and 3% KOH solution (Bandoni 1979). Meanwhile, PCR amplification and DNA sequencing of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) was performed by using the universal primers ITS1/ITS4 (White et al. 1990) and the AG2-2IIIB specific primers (see Supplementary Fig. 1C for PCR results and primer sequences) described by Carling et al. (2002). The resulting sequences that were amplified with the ITS1/ITS4 primers (GenBank accession no. MT258822) and the AG2-2IIIB specific primers (GenBank accession no. MT679722) showed more than 98% and 100% identity to those of more than 10 AG-2-2IIIB isolates present in GenBank, respectively. Based on the morphological characteristics and molecular analysis, the isolate was identified as R. solani AG-2-2IIIB. Koch's postulates were carried out to prove the pathogenicity of the isolate. First, disease-free minitubers (cv. Favorita) of approximately the same size (10-20 g) were allowed to sprout at room temperature for 10 days. The minitubers were then planted into 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 by 7-mm-diameter mycelial plugs (from one PDA petri plate) over the minituber, which was then covered with potting mix. The control pots were inoculated with sterile PDA. Each treatment consisted of 10 plants, and the experiment was repeated three times. After 3 weeks, plants were removed and assessed for disease. Typical stem cankers were observed on plants grown in pots inoculated the mycelial plug, but the control plants remained disease free. Moreover, R. solani AG-2-2IIIB was reisolated consistently from symptomatic stems, and the identity of the reisolates were confirmed by the above mentioned morphological and molecular analyses. To our knowledge, this is the first report of AG-2-2IIIB causing disease on potatoes in Heilongjiang Province, the main potato-producing area of China. Potato black scurf caused by R. solani AG-2-2IIIB has been reported in Inner Mongolia Autonomous Region (Tian et al. 2011). Although it is not considered a major potato disease in Heilongjiang Province, with the recent rapid development of the potato industry, it can become a major problem due to the high risk of soil borne infection and disease dispersal with regard to seed potato transportation across China. The information presented in this note will assist in developing management strategies for this disease.