**4.1 Round spot disease occurrence characteristics**

We had observed the disease from 2016 in the *P. notoginseng* plantations in Lancang County, Pu′er City, Yunnan Province. In rainy season, round, water-soaked spots on the leaves of *P. notoginseng* appeared when it was exposed in rain. Round or nearly round watery spots on leaves are initial symptoms of round spot disease. The lesion appeared transparent when it was viewed in the sun compared with healthy leaf tissue. Then the brown infection spots were observed in the center of the disease spots (**Figure 3A**). Eventually, the lesions gradually expanded, turned brown or grayish brown, and had "wheel marks" (**Figure 3B**). Later, white powdery conidial piles formed on the lesion surface (**Figure 3C**). Finally, multiple lesions merged, causing

#### **Figure 3.**

*Symptoms, incidence, and disease index of round spot disease. (A) Watery spots on* P. notoginseng *leaves. (B) Watery spots expand to form black wheel-like lesions. (C) Gray white conidia piles form on the lesions. (D) Leaves fall off in the late stage of the disease. (E) Relationship between incidence rate of round spot disease and monthly rainfall in DTZ and XGZ. (F) Relationship between disease index of round spot disease and monthly rainfall in DTZ and XGZ. XGZ: Xiaoguangzha* P. notoginseng *plantation. DTZ: Datangzi*  P. notoginseng *plantation.*

the leaves to rot and fall off during rainfall (**Figure 3D**). Based on these characteristics of the disease spots, the disease was preliminarily identified as round spot disease.

The incidence of round spot disease in Lancang County was investigated and monitored. *P. notoginseng* begins to germinate from March to April. From March to June, no round spot disease symptoms were observed (**Figure 3E** and **F**). As of July, round spot disease started to occur. Round spot disease incidence rose in August and September. Lancang County has a subtropical wet summer and dry winter mountain monsoon climate, with distinct rainy and dry seasons. Generally, rainfall is limited in April and May, but begins to increase as of June. **Figure 3E** and **F** shows that round spot disease started to occur after the rainy season.

#### **4.2 Isolation and identification of the causal pathogen of round spot disease**

The pathogen causing round spot disease was isolated by single-spore isolation. In total, 30 pathogen strains were isolated from 30 diseased leaves. On PDA, colonies were round, villous, and varied in color from red to gray (**Figure 4A** and **B**). The strains did not produce conidia on PDA; they required water to induce conidia production. Conidiophores produced by thickened hyphae were colorless and bent (**Figure 4F** and **G**). Conidia were solitary, colorless, transparent, and long-arched, with multiple diaphragms (**Figure 4C** and **D**). The tail of the conidia gradually became thinner and the apical cells appeared to be truncated. A long, narrow, accessory filament with septa grew on the side of the apical cell. Notably, thin cilia were observed at the tail of the conidium and the top of the accessory filament (**Figure 4E**). The total length of conidia, including the accessory filament and two cilia, was 171.83–492.92 μm (average, 349.74 μm) (n = 200). Their width, measured at the widest part in the middle of the conidia, was 6.24–13.05 μm (average,

#### **Figure 4.**

*Morphological characteristics of* Mycocentrospora acerina*. (A, B) mycelial colony morphology of*  M. acerina *cultured on PDA medium for 7 days at 20°C. (C–E) morphological characteristics of conidia. (F, G) morphological characteristics of conidiophores and conidia. The conidiophores and conidia in panel G were stained with calcofluor white. c: Conidiophore. (H) Conidia on the leaves of* P. notoginseng *germinated to produce an appressorium and penetration peg. A: Appressorium, pp.: Penetration peg. (I) Symptoms on*  P. notoginseng *leaves 7 days after inoculation of a conidial suspension. (J) Symptoms on* P. notoginseng *leaves 14 days after inoculation of a conidial suspension. (K)* P. notoginseng *leaves remained healthy for 7 days when being mock-inoculated with sterile water. Scale bars = 10 mm in A–B, scale bars = 50 μm in C–H.*

8.61 μm). Based on the morphology and conidial characteristics, the pathogen was identified as *M. acerina* [47].

The pathogenicity of 10 isolated strains was determined. Seven days post inoculation, all inoculated leaves showed similar lesions, and the symptoms were identical to those in the field (**Figure 4I** and **J**). The control plants remained healthy (**Figure 4K**). The diseased leaves in each treatment were selected for pathogen isolation, and the pathogen was reidentified as *M. acerina*.
