Patchouli (Pogostemon cablin Benth.) is a perennial herb native to South and South East Asia, which widely used as a traditional herbal medicine against indigestion, diarrhea, cold, fever, vomiting and headache in China (Swamy and Sinniah, 2015). In September 2020, a new basal stem and root rot disease of patchouli was observed in three cultivated fields of Zhanjiang City, Guangdong Province. Symptoms included sudden discoloration, chlorosis and wilting of the leaves and severe rot associated with external and internal browning at the basal part of stems and roots that results in the death of approximately 2 to 5% of plants in each field. To determine the causal agent, symptomatic roots with typical lesions were cut into small pieces, then surface sterilized in 2.5% NaClO for 1 min, rinsed three times in distilled water, and then inoculated on potato dextrose agar (PDA) medium. A fungus with same morphological characteristics was consistently isolated from disease tissue. The mycelia initially white to cream, later turning pale brown to black with age on PDA. To induce the sporulation, the isolated fungi were transferred to synthetic nutrient-poor agar (SNA) with autoclaved pine needles and ten pure cultures were obtained by single spores. Masses of black, hard and oblong microsclerotia (av. 171.2×136.5 µm, n=50) were observed on SNA. Conidia hyaline, ellipsoid to obovoid, smooth, enclosed in a mucous sheath, 15.9~32.5×7.7~11.9 µm (av. 23.9 × 9.6 µm, n=100). The internal transcribed spacer (ITS) regions as well as the partial translation elongation factor (EF-1α) and β-tubulin (TUB) genes of two representative isolates GHX-1 and CHX-2 were sequenced, using primer pairs ITS1/ITS4 (White et al. 1990), EF-688F/EF-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The obtained sequences were submitted to GenBank under the following accession numbers: ITS, MZ375360 and MZ375361; EF-1α, MZ393804 and MZ393805; and TUB, MZ393806 and MZ393807. BLASTn searches revealed that 100% identity with the existing sequences of ex-type culture CGMCC3.19503 of Macrophomina vaccinii (ITS, MK687450; EF-1α, MK687426; and TUB, MK687434), respectively. Phylogenetic analysis using Neighbour-Joining method in Mega7.0 with concatenated sequences of ITS, EF-1α and TUB showed that the isolates clustered in the clade of M. vaccinii with high bootstrap support values. Based on both of the morphological and molecular results, the isolates were identified as M. vaccinii (Zhao et al., 2019). To confirm the pathogenicity of the two isolates, 5-mm-diameter mycelial agar plus from the margins of 5-day-old PDA cultures were placed on the wounded basal stem of 2-month-old patchouli seedlings in each pot. Five separate pots were used for each isolate and plants inoculated with sterile agar plus were served as controls. All plants were covered with plastic bags to maintain 90% relative humidity and kept at 26°C with a photoperiod of 12 h in a greenhouse conditions. The patchouli plants developed similar necrotic symptoms on basal stem and root to those observed in the field after 3 weeks after inoculation, whereas the control plants were asymptomatic. M. vaccinii was reisolated from inoculated plants and identify by morphological and molecular characteristics, fulfill the Koch’s postulates and identify. Recently, stem blight on blueberry caused by M. vaccinii was reported in China (Zhao et al. 2019). To our knowledge, this is the first report of M. vaccinii causing basal stem and root rot on patchouli in China and worldwide.
Morphology, pathogenicity, and molecular identification of Fusarium solani species complex (FSSC) associated with potato tubers
