Systemic Colonization of Potato Plants by Verticillium dahliae Leads to Infection of Tubers and Sprouting Buds.

A green fluorescent protein (GFP)-tagged isolate of Verticillium dahliae was used to study its colonization in potato plants and tubers. Three-week-old potato plants of the highly susceptible cultivar 'Shepody' were inoculated with a conidial suspension of a GFP-tagged isolate of V. dahliae using a wound inoculation method. Colonization was studied using confocal microscopy combined with tissue sections. Conidia germinated and hyphae grew along the root hairs, elongation zones, and root caps between 24 and 96 h postinoculation (HPI). At 7 days postinoculation (DPI), the pathogen advanced to cortical tissues and grew into the root vascular bundles. At 8 weeks postinoculation (WPI), the stem epidermal cells, cortical tissues, vascular elements, and petioles were fully colonized by the mycelium of V. dahliae. At 11 WPI, the pathogen was detected in the stolon and progeny tubers, as confirmed by both GFP signals in tissues and reisolation of the pathogen on the semiselective NP-10 medium. Progeny potato tubers were harvested from the inoculated potato plants, and the GFP-signal was observed in the epidermal cells and vascular elements of sprouting buds that emerged from the harvested tubers. The infection rate of progeny tubers detected on semiselective NP-10 medium ranged from 34.55 to 55.56%, with an average of 45.31%. In conclusion, we report, for the first time, the entire progression of colonization by V. dahliae in potato plant tissues, progeny tubers, as well as of the sprouting buds that emerged from progeny tubers.

First Report of Chinese Cabbage Wilt Caused by Plectosphaerella cucumerina in Inner Mongolia of China.

Chinese cabbage [Brassica rapa L. subsp. pekinensis (Lour.) Hanelt] has been grown commercially for many decades in Huade County, Inner Mongolia. In 2018 and 2019, an unusual stem and leaf wilt disease with an average disease incidence of approximately 3% was observed. Diseased plants with spindle-shaped stem lesions were collected and small pieces (0.3 × 0.3 cm) of the diseased tissues were cut from the margins of stem lesions, surface sterilized with 75% alcohol for 3 to 5 s, 0.1% NaClO for 2 to 3 min, and washed three times with sterilized water. The treated tissues were placed on 1.5% (w/v) water agar plates and incubated at 25°C for 3 days. The mycelia were cut and transferred onto potato dextrose agar (PDA) for culture purification. Three isolates with similar morphology were obtained and named as BC-2, BC3-2 and BG2. To confirm their pathogenicity, Chinese cabbage (cv. Chunqiuhuang) seed was planted into plugs. After 30 days, the fibrous roots were wounded with a fruit knife and root-dipped in the conidium suspension (1 × 106 conidia/ml) for 20 min. Inoculated seedlings were transplanted in pots (30 × 25 cm) with sterilized nursery soil, with one seeding per pot. The roots of control plants were also wounded and dipped in sterilized water. Five seedlings were inoculated with each isolate and the experiment was repeated three times. Treated seedlings were maintained in a greenhouse at 25 to 28°C under a 12-h photoperiod. Chlorosis and wilting were observed approximately 4 weeks after inoculation, and the outer layer of leaves of the inoculated seedlings developed discoloration and wilted symptoms after 50 days. The symptoms induced by all three isolates were the same as the symptoms observed in the field, whereas no symptoms developed on the control plants. To confirm the Koch's postulates, the fungus was successfully re-isolated from the infected leaves and had similar growth and morphology as the original isolates. The three isolates were cultured for both morphology and molecular identification. The 14-day-old colonies on PDA were buff or salmon pink with few aerial hyphae, and slimy surfaces. Aerial hyphae were sparse with simple or branched conidiophores. Conidia were ellipsoidal, hyaline, surface smooth, septate or aseptate, and (4.0 to 9.7 µm × 2.0 to 3.9 µm). Such characteristics are typical of Plectosphaerella spp. (Palm et al. 1995). For molecular identification, the internal transcribed spacer (ITS) region of rDNA was amplified using the primer pair ITS1/ITS4 (White et al. 1990), and the products were directly sequenced. BLAST analysis showed that the sequences of Isolates BC-2 (511 bp out of 515 bp), BC3-2 (512 bp out of 516 bp) and BG2 (503 bp out of 505 bp) showed 99% identity to an isolate of P. cucumerina (acc. no. KT826571.1) from Bottle gourd [Lagenaria siceraria (Molina) Standl.] (Yan et al. 2016). The sequences of Isolates BC-2, BC3-2 and BG2 were deposited in GenBank (acc. nos. MW320463, MW320462 and MW320464). Although P. cucumerina was reported causing root rot of cabbage (B. oleracea) in Gansu, China (Li et al. 2017), to our knowledge, this is the first report of P. cucumerina causing Chinese cabbage wilt in Inner Mongolia, China. The presence of the disease could cause significant economic losses in Chinese cabbage production. For this reason, strategies for the management and control of this disease should be implemented.

Identification and comparison of biological characteristics and pathogenicity of different mating types of V. dahliae isolated from potato and sunflower.

Potato is one of the most important staple crops in the world. China is one of the leading producers of potatoes, but the industry faces soilborne diseases such as Verticillium wilt. Most potato planting areas in China rotate the crop with sunflower which is also highly susceptible to Verticillium wilt. The comparison of the biological characteristics and pathogenicity of different mating types of Verticillium dahliae isolated from potato and sunflower in the major planting regions in China is of great importance. This is to help unravel the diversity in V. dahliae population and the sudden increase in infected fields. The diseased samples collected were cultured on PDA and the growing colony of pathogen isolated. Molecular techniques using specific primers were used to identify the V. dahliae pathogens and their mating type of the isolates obtained from the diseased sunflower and potato plants as well as their planting materials. The data obtained revealed that the dominant mating type population in sunflower was MAT1-1, whiles that of potato was MAT1-2, but Race 2 was the only race type identified for all the samples. There was a significant presence of MAT1-1 isolates present in potatoes, which is a new trend. Conventional crop rotation farming using sunflower is causing an increasing prevalence of MAT1-1 and mating type shift of isolates in potato in these regions.

Genome-wide screening and identification of nuclear Factor-Y family genes and exploration their function on regulating abiotic and biotic stress in potato (Solanum tuberosum L.).

The Nuclear Factor-Y (NF-Y) transcription factor (TF), which includes three distinct subunits (NF-YA, NF-YB and NF-YC), is known to manipulate various aspects of plant growth, development, and stress responses. Although the NF-Y gene family was well studied in many species, little is known about their functions in potato. In this study, a total of 37 potato NF-Y genes were identified, including 11 StNF-YAs, 20 StNF-YBs, and 6 StNF-YCs. The genetic features of these StNF-Y genes were investigated by comparing their evolutionary relationship, intron/exon organization and motif distribution pattern. Multiple alignments showed that all StNF-Y proteins possessed clearly conserved core regions that were flanked by non-conserved sequences. Gene duplication analysis indicated that nine StNF-Y genes were subjected to tandem duplication and eight StNF-Ys arose from segmental duplication events. Synteny analysis suggested that most StNF-Y genes (33 of 37) were orthologous to potato's close relative tomato (Solanum lycopersicum L.). Tissue-specific expression of the StNF-Y genes suggested their potential roles in controlling potato growth and development. The role of StNF-Ys in regulating potato responses to abiotic stress (ABA, drought and salinity) was also confirmed: twelve StNF-Y genes were up-regulated and another two were down-regulated under different abiotic treatments. In addition, genes responded differently to pathogen challenges, suggesting that StNF-Y genes may play distinct roles under certain biotic stress. In summary, insights into the evolution of NF-Y family members and their functions in potato development and stress responses are provided.

Small G Protein StRab5b positively regulates potato resistance to Phytophthora infestans.

Rabproteins are the largest members of the small G protein family and are widely distributed in eukaryotes. It comprises eight subfamilies and is responsible for regulating vesicle transport, plant growth and development, and biotic and abiotic stress responses. In this study, the small G protein gene StRab5b was cloned from potato, and its biological information, expression profile and induced expression level, overexpression and gene silencing were examined on regulating potato resistance to Phytophthora infestans using PCR, qPCR and Virus-induced gene silencing (VIGS). Our results indicate that the amino acid of StRab5b shows the highest and lowest homology with NbRab5b in N. benthamiana and StRab in potato respectively. StRab5b expression varied among different potato tissues and varieties, and was induced by P. infestans infection. Transiently ectopic expression of StRab5b in N. benthamiana enhanced its resistance to P. infestans, whereas, silencing of StRab5b and its homologous gene facilitated pathogen infection in potato and N. benthamiana respectively. Furthermore, stable expression of the StRab5b gene in potatoes enhanced its redox-stress response capacity, as manifested by the accumulation of H2O2 in infected leaves and subsequent increase in the activity and expression of ROS scavenging enzymes, thereby attenuating the development of P. infestans and ultimately reducing the lesions on infected potato leaves. In addition, the LOX gene transcripts and JA level were upregulated rapidly after inoculation with P. infestans. Collectively, our results suggest that StRab5b positively regulates the resistance against potato late blight (PLB) via JA-mediated defense signaling pathway.

The α-1,6-mannosyltransferase VdOCH1 plays a major role in microsclerotium formation and virulence in the soil-borne pathogen Verticillium dahliae.

Sunflower yellow wilt is a widespread and destructive disease caused by the soil-borne pathogen Verticillium dahliae (V. dahliae). To better understand the pathogenesis mechanism of V. dahliae in sunflower, T-DNA insertion library was generated via Agrobacterium tumefaciens mediated transformation system (ATMT). Eight hundred positive transformants were obtained. Transformants varied in colony morphology, growth rate, conidia production and pathogenicity in sunflower compared to the wild type strain. A mutant, named VdGn3-L2, was chosen for further analysis based on its deprivation on microsclerotia formation. The flanking sequence of T-DNA insertion site of VdGn3-L2 was identified via hiTAIL-PCR, and the interrupted gene encoded an initiation-specific α-1, 6-mannosyltransferase, named as VdOCH1. The deletion mutant ΔVdOCH1 was impaired in certain characteristics such as fungal growth, conidia production, and microsclerotia formation. Also, ΔVdOCH1 mutants were more sensitive to the cell wall perturbing reagents, such as SDS and Congo red, lost their penetration ability through cellophane membrane, and exhibited dramatically decreased pathogenicity to sunflower. The impaired phenotypes could be restored to the wild type level by complementation of the deletion mutant with full-length VdOCH1 gene. In conclusion, VdOCH1, encoded α-1,6-mannosyltransferase, manipulating the biological characteristics, microsclerotia formation and pathogenic ability of V. dahliae in sunflower.