Wilting of branches and leaves was observed on 4-5 year old apple trees of the varieties Delicious and Fuji in orchards located in Wushan, Gansu Province, China in April 2018. Subsequently, the stem vascular tissue and woody xylem became discolored and necrotic. The stem dieback expanded rapidly to the entire vasculature of the branches. Finally, the epidermis of the stem bases split and was covered with light pink mold. For the pathogen isolation, 25 symptomatic stems were collected from 25 symptomatic trees in 3 individual orchards. Fragments (approximately 0.5 cm in length × 0.5 cm in width) of symptomatic stems were surface sterilized and individually transferred to Petri dishes containing potato dextrose agar (PDA), and incubated for 4 days at 25°C. Five types of isolates with distinct morphological characteristics (PJ1 to PJ5) were obtained from the 25 symptomatic stems after the single spore inoculation and sub-culture. The isolation frequency of PJ1, PJ2, PJ3, PJ4 and PJ5 types was 11%, 8%, 100%, 4% and 13%, respectively, in the 25 symptomatic stems. A spore suspension of PJ1, PJ2, PJ3, PJ4 and PJ5 types was prepared by adding 5 ml of sterile distilled water in the 14-day old culture colonies and filtered through 0.22 mm Millipore membranes, and the final concentration was adjusted to 108 per ml for inoculation. Detached healthy apple stems (15 cm in length) were surface-disinfested and used to evaluate the pathogenicity of PJ1 (7 isolates), PJ2 (5 isolates), PJ3 (32 isolates), PJ4 (2 isolates) and PJ5 (9 isolates) by dipping the stems into sterilised tubs containing the spore suspension (108 per ml) of each isolate. Apple stems dipped in sterile distilled water served as the control. Each control and treatment were repeated 3 times. At day 15 and 35, the stems infected with the spore suspension of PJ3 isolates developed symptoms that were similar to those observed in the apple orchards. However, the other four types (PJ1, PJ2, PJ4 and PJ5) exhibited either no symptoms or different symptoms from those observed in the apple orchards. There were no symptoms on the control stems. After the colony of the pathogen (PJ3 type) was re-isolated from the infected stem bases 35 days inoculation. The PJ3 type isolates with same morphological characteristics as the original PJ3 type isolates were used for further examination and identification. After 4 days of incubation on PDA, the colonies of PJ3 type isolates developed velvety aerial mycelia with white or light pink color when they were viewed from the front/top side of the PDA and orange-red color when they were viewed from the reverse/bottom side. After 14 days of incubation, the color in the centre of the colonies changed to yellow green in the front view and carmine red in the reverse view of the plates. Three types of spores (microconidia, macroconidia and chlamydospores) were observed after incubation of PJ3 type isolates for 14 days. The size (width and length) of 30 conidia in each of PJ3 type isolates was measured and averaged. The microconidia were abundant on aerial mycelia and limoniform, oval or pyriform with 0-1 septa. Their size ranged from 1.94 μm to 8.05 μm in length and 1.48 μm to 3.62 μm in width. The macroconidia were falciform and curved in shape, mostly with 3-5 septa and a size ranging from 13.52 μm to 22.43 μm in length and 2.31 μm to 3.87 μm in width. The chlamydospores were spherical, intercalary and formed in chains on PDA plates. These morphological characteristics indicate that the PJ3 type isolates could be Fusarium tricinctum (Chen et al. 2019; Aktaruzzaman et al. 2018). To confirm the morphological identification, the sequences of internal transcribed spacer (ITS), transcriptional enhancer factor-1 (TEF-lα) and ribosomal RNA large subunit gene (LSU) of the representative isolate PJ3-3 selected from the PJ3 type isolates with same morphological characteristics were sequenced and used for molecular identification (Laurence et al. 2011; Abd-Elsalam et al. 2003; Miller et al. 1996). The sequences of ITS, TEF-lα and LSU of the PJ3-3 isolate were deposited in NCBI database with the accession numbers of MZ799356, MZ820045 and MZ820044, respectively. In BLAST analyses, the obtained sequences of the PJ3-3 isolate showed 99.47%, 100% and 99.01% identity to the corresponding region of F. tricinctum ITS (JX179207.1: 3-566 Fusarium tricinctum isolate Fyx 1), TEF-lα (MK032320.1 F. tricinctum isolate ZD3) and LSU (KC311496.1 Fusarium tricinctum isolate L12), respectively. The phylogenetic analysis clustered the PJ3-3 isolate sequences within the same clade with ITS, TEF-lα and LSU sequences of F. tricinctum isolates. Thus, the PJ3-3 isolate was identified as F. tricinctum based on the pathogenicity tests, morphological characteristics and molecular analyses. Previously, the symptoms of xylem browning and dieback were observed in the twigs of wild apple trees that were collected from wild apple forests, and the species F. avenaceum, F. solani, F. tricinctum, F. proliferatum, and F. sporotrichioides were isolated from diseased wild apple trees (Chen et al. 2019). Only F. avenaceum, F. solani, F. proliferatum, and F. sporotrichioides were reported as the pathogens causing the disease symptoms of xylem browning and dieback in wild apple trees in Xinjiang, China (Chen et al. 2019). In our present study, we found that F. tricinctum can cause stem vascular and woody xylem browning, wilting, and dieback in the apple tree varieties Delicious and Fuji. These are new symptoms discovered in our present research and different from the previous paper (Chen et al. 2019). Therefore, to our knowledge, this study is the first report of F. tricinctum causing a new disease on apple trees in China following Koch’s postulates. Our findings are important for the management of apple disease and protect apple trees in the future.
Pesticide exposure of workers in apple growing in France
