Pinus thunbergii Parl. (Japanese black pine), an evergreen species, is distributed along the seacoasts of China. In addition, this species has been planted along seacoasts as a windbreak to prevent soil erosion due to its resistance to salt and various environmental stresses. It can also be found in public parks and gardens due to its exquisite appearance and toughness. In August 2020, needle blight symptoms were found on several black pine trees in Sichuan Province, China. Further surveys showed that these symptoms are common. The disease incidence is less than 30% while severity of the disease is high. The tips of old needles first turn grayish green that developed into brown bands ranging from 1 to 2 mm. To determine the pathogen, small needle pieces (3-4 mm2 long) from the margin of fresh lesions were surface-sterilized for 30 s in 75% ethanol, follow by 1% NaOCl for 90 s, then washed three times with sterile water, and then were placed on potato dextrose agar (PDA) with 0.1 mg/mL ampicillin and incubated at 25°C. Pure cultures of 8 isolates were obtained by monosporic isolation, and a representative isolate (SC03) was deposited in the Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University. When cultured on Spezieller Nährstoffarmer Agar medium (Leslie and Summerell, 2006), the SC03 colony was beige-white, cottony from top view and pale orange near the center on the reverse side. The daily growth rate was 11.8 mm/day at 25°C in the dark. Microscopic observations showed hyaline and septate hyphae, slightly curved macroconidia with two to three septa measuring 17.5 - 30 × 3.7 - 7.5 μm (23.2 × 5.7 on average), and aseptate microconidia measuring 7.5 - 12.5 × 2.5 - 5.0 μm, (9.7 × 4.3 on average). The morphological characteristics of conidia and other structures of SC03 matched those of the Fusarium fujikuroi species complex (FFSC) (Abdalla et al. 2000). For accurate identification, translation elongation factor 1-alpha（TEF-1α） , and the second largest RNA polymerase subunit (RPB2) were amplified and sequenced using the primer pairs EF1 and EF2, RPB5f2 and RPB7cr. The sequences were deposited in GenBank [Accession TEF-1α: ON049647, RBP2: ON049648]. A Blast search of GenBank showed that TEF-1α and RPB2 sequences of SC03 matched Fusarium proliferatum (Matsush) Nirenberg at a high level (>99%). Phylogenetic analysis using neighbor joining and concatenated sequences (TEF-1α and RPB2) with MEGA X placed SC03 in F. proliferatum. For the pathogenicity test, a conidial suspension was prepared with a concentration of 2.0 × 107 conidia/ml. The suspension was sprayed onto 3 annual seedlings' needles, and the control was sprayed with sterile water. Inoculated and uninoculated plants were kept in humid chambers in a glasshouse where the average humidity was 60% and the average temperature was 27℃. After 10 days, typical symptoms appeared on inoculated needles, whereas control needles remained symptomless. These symptoms were similar to those observed in field. The fungus, F. proliferatum, was reisolated from those lesions, confirming Koch's postulates. No symptoms were observed on control plants. Fusarium proliferatum is a ubiquitous saprophytic fungus on cankers and very rarely reported to cause disease on pine needles. It has been reported to cause leaf blight of Polygonatum cyrtonema (Zhou et al. 2021) and Majesty palm (Polizzi and Vitale 2003). To our knowledge, this is the first report of needle blight on P. thunbergii caused by F. proliferatum in China. The disease represents a threat to producers and more research on the biology and management is needed.
Keywords: Causal Agent; Crop Type; Fungi; Pathogen detection; Subject Areas; Trees; forest.