First Report of <i>Penicillium citreonigrum</i> Causing Root Rot Disease in Blueberries in China

Blueberry (Vaccinium corymbosum) plants are small, berry-producing shrubs belonging to the family Ericaceae. On July 24, 2024, root rot symptoms were observed in Huaining, Anhui Province, China (30°44′12.8″N, 116°49′28.4″E). Similar symptoms were observed in approximately 5% of 420 blueberry seedlings cultivated in substrate containers under greenhouse conditions. Affected plants exhibited leaf yellowing, curling, wilting, and eventually defoliation. Symptomatic roots were harvested and washed with sterile water, surface-sterilized twice using 75% ethanol for 30 s, and rinsed 3–4 times with sterile water. The roots were placed on potato dextrose agar (PDA) medium and incubated at 25°C for 7 days. Three isolates (HN-Pc1, HN-Pc2, and HN-Pc3) exhibiting similar morphology were obtained using single-spore isolation. The isolates produced aerial mycelium that appeared white to pale gray on the front view and light yellow on the reverse view. After 7 days of incubation, colonies reached a diameter of 29 mm. The conidia were single-spored, spherical or ellipsoid grayish green (2.56 ± 0.36 μm in diameter, n = 50). Conidiophores produced broom-like conidiosphores with multiple branches bearing phialides. These morphological characteristics were similar to those described for P. citreonigrum, P. citreosulfuratum, and P. fundyense (Hu et al. 2021; Visagie et al. 2016). To further confirm species identity, the conserved regions of RNA polymerase II second-largest subunit (RPB2), β-tubulin (TUB2), and calmodulin (CaM) genes were amplified using primer pairs RPB1-5f/RPB2-7cR, Bt2a/Bt2b, and CMD5/CMD6, respectively (Visagie et al. 2014). The resulting RPB2, TUB2, and CaM sequences were deposited in GenBank under accession Nos. PV593986, PQ887759, and PQ887758, respectively. In a BLASTn search, the CaM, TUB2, and RPB2 sequences showed 96.11%, 98.25%, and 98.27% similarity to P. citreonigrum strains in the NCBI database (accession numbers EF198581, OU641462, EF198500). Multiple-locus (RPB2–TUB2–CaM) alignment confirmed the highest similarity to P. citreonigrum NRRL 761 and NRRL 1187. To fulfill Koch’s postulates, inoculation trials were conducted using healthy ‘O’ Neal’ blueberry plants (Vaccinium corymbosum). Each plant received a P. citreonigrum HN-Pc1 spore suspension (25 mL, 1 × 10⁶ conidia/mL) through root irrigation. In parallel, control ‘O’ Neal’ plants were treated with an equivalent volume of sterile water. All treated plants were maintained at 25°C for 7 days. The inoculated plants developed symptoms identical to field observations and root discoloration (brown to dark brown). In contrast, no symptoms were observed in the control plants. The fungus was re-isolated and identified as P. citreonigrum based on morphological characteristics and conserved multiple sequence alignment analysis (RPB2–TUB2–CaM) (Visagie et al. 2016). To the best of our knowledge, this is the first report of root rot disease caused by P. citreonigrum in blueberry plants in China. These findings provide a basis for the early detection of P. citreonigrum, and lay a crucial foundation for developing effective management strategies.