First Report of Fusarium oxysporum and Fusarium solani Causing Root Rot on Trifoliate Orange Rootstock in China

Trifoliate orange (Poncirus trifoliata L) is a thorny tree of the rue family that is extensively used as a citrus rootstock in China. In January 2021, several leaf yellowing, declining, and wilting citrus seedlings grafted on trifoliate orange rootstock with rotted main roots were observed in orchards in Wuhan city, Hubei, China. In old orchards, the incidence of diseased roots was approximately 90%. Diseased roots from seven plants were collected and cut into small pieces (0.2 to 0.5 cm). These pieces were surface sterilized using 0.1% mercury bichloride for 3 min and 75% ethanol for 3 min, rinsed with sterile distilled water several times, placed on potato dextrose agar (PDA) supplemented with 0.05% lactic acid (v/v), and incubated at 25 ± 2°C in the dark. Fifty-three single-conidium isolates with morphological characteristics similar to Fusarium spp. were obtained (Leslie and Summerell 2006), which displayed two kinds of colony morphology. Thirty isolates showed white to orange-white abundant aerial mycelium in rings and acquired a yellow to orange pigmentation, and 23 isolates showed white to pink, fluffy aerial mycelium in rings and acquired an orange to red pigmentation. Isolate WG-1 and HrmY-9 from each group were used for further identification. The average colony growth rates of WG-1 and HrmY-9 on PDA was 0.95 ± 0.06 and 0.69 ± 0.11 cm/day (n = 4), respectively. WG-1 produced numerous oval, unicellular microconidia without septa, 4.03 to 9.87 × 1.01 to 5.13 μm (n = 80), and very few macroconidia with two to four septa, narrowed at both ends, 11.08 to 22.64 × 1.67 to 4.91 μm (n = 30). HrmY-9 produced numerous curved macroconidia with three to four septa, 18.03 to 37.33 × 2.16 to 7.8 μm (n = 80), microconidia were unicellular, oval, and 5.33 to 16.19 × 1.74 to 6.51 μm (n = 50). Sequences of internal transcribed spacer (ITS), translation elongation factor 1-alpha (EF-1α) gene, and DNA-directed RNA polymerase largest subunit (RPB1) gene were amplified with the primers ITS1/ITS4, EF1a-F/EF1a-R, and RPB1-F5/RPB1-R8, respectively (O’Donnell et al. 1998, 2010; White et al. 1990), sequenced, and deposited in GenBank. Sequences of isolate WG-1 (accession nos. ON045437, ON063232, and ON089664) and HrmY-9 (accession nos. ON045438, ON063233, and ON089665) were 100% identical with corresponding sequences of Fusarium oxysporum (OM876904, JF430180, and MT568959) and F. solani (MT605584, MK617767, and MT305110), respectively. Based on these results, WG-1 and HrmY-9 was identified as F. oxysporum and F. solani, respectively. Pathogenicity tests were performed on healthy 1-year-old trifoliate orange seedlings by dipping their injured roots into conidial suspension (50 ml, 1 × 10⁶ conidia/ml) for 1 h and the rest of the conidial suspension was added to the pot after replanting to make sure the inoculum was in contact with the roots. Roots of control plants were inoculated with sterilized water. All experiments were repeated twice. All plants were cultured at 26°C under a 16-h light/dark cycle. Typical symptoms developed on most inoculated seedlings 2 months postinoculation. No disease symptoms appeared on control plants. The same colonies were reisolated from the inoculated roots, confirming Koch’s postulates. To our knowledge, this is the first report of F. oxysporum and F. solani causing root rot on trifoliate orange rootstock in China. The identification of F. oxysporum and F. solani as the causal agents of the observed root rot on trifoliate orange rootstock is critical to the prevention and control of this disease in the future.