2.1 Sample collection and pathogen isolation

The plant and rhizosphere soil samples of infected R. lapponicum were collected at a plantation area in Anning City (24°55′08″N, 102°28′41″E), Yunnan province, China in January 2021. Samples were packed in polythene bags and brought to the laboratory for further analyses. Root samples were washed with tap water and surface-sterilized with 75% ethanol for 30 s and further washed with ddH₂O three times. Roots were cut into 0.5 cm pieces with sterilized scissors and placed on potato dextrose agar (PDA) and oatmeal agar (OA) medium, containing rifampicin and ampicillin (30 mg/L each) to avoid bacterial contamination, followed by incubation at 28°C for 3–5 days. The mycelium discs at the edge of typical colonies were selected and transferred to the new media plates for purification and cultivation. Pure culture was transferred into the PDA and OA medium glass tubes and added with sterilized paraffin oil after the mycelium grow over the whole slant, then the tubes were stored at 16°C for further use.

2.2 Pathogenicity test

To better understand the pathogenicity of isolated strain, we used healthy R. lapponicum plants as inoculating hosts. The pathogen was cultured for 7 days in a PDA plate (several plates) and the suspension was prepared by adding 50 ml of sterile distilled water. The roots of healthy R. lapponicum were drenched with 20 ml of mycelium suspension through soil. The sterile water was used as a control. Furthermore, all inoculated plants were transferred to the greenhouse (28°C, 75% relative humidity) to observe the disease symptoms. After 45 days of inoculation, the plants inoculated with mycelium suspension were observed for symptoms of leaf yellowing, withered and drooped, and rotten roots. A total of 15 pots were used for these experiments with three biological replicates. The diseased root samples were collected for pathogen confirmation.

2.3 Morphological observation and molecular identification

The candidate pathogen was cultured in an OA medium with 0.1% potassium nitrate at 28°C for 7 days. The plates were kept for 12 h in light and dark each at 28°C for 5 days to induce chlamydospore and sporangium. The morphology of mycelium and spores were observed under a light microscope (Carl Zeiss Microlmaging GmbH 37081). The pathogen was identified and the taxonomic status of the isolate was preliminarily clarified. To get the exact identification of pathogenic isolate, molecular analyses were carried out.

The genomic DNA of the isolate was extracted using cetyl trimethylammonium bromide (CTAB) method [12]. The primer Btub-F1/Btub-R1 was used to amplify the β-tubulin gene [13] and ITS1/ITS4 primers were used to amplify the sequence of internal transcriptional spacer (ITS) of ribosomes [14]. Primer Yph1F/Yph2R amplified the encoding gene of Ypt1 associated with the Ras family of Phytophthora [15]. List of all primers is mentioned in Table 1. The PCR amplification products were sequenced by Shanghai Qingke Biotechnology Co., Ltd. and the sequencing results were compared with the homologous sequence information BLAST (basic local alignment search tool) in GenBank (http://www.ncbi.nlm.nih.gov/balst/) and Phytophthora ID (http://phytophthora-id.org/index.html). Phylogenetic trees were built through MEGA 6 using the neighbor-joining method.

2.4 Host range determination

Host range of this pathogen was determined using more than 60 types of common landscape plants and fruit trees and R. lapponicum was used as a positive control. The leaves of tested plants were collected and inoculated with a 7 mm mycelium disc of pathogenic isolate and cultured in a PDA plate with absorbent paper with three replications. All leaves were stored in a 28°C constant temperature light incubator (12 h in light and in dark each, 4000 lux of light), to observe and record disease incidence.

2.5 Biological characteristics of pathogen

2.6 Screening and identification of potential antagonistic strains

One gram of root tissue and rhizosphere soil of healthy R. lapponicum plant were macerated in sterile mortar and pestle to make different suspension to spread on Luria–Bertani (LB) and PDA plates, respectively. The LB plates were incubated at 37°C for 2 days in the dark and single colonies were picked for marking and preservation. Accordingly, PDA plates were incubated at 28°C for 2–7 days in the dark. The emerging colonies of fungi were picked, purified, and preserved. The candidate bacteria isolated in this experiment were used as indicators. Antagonistic activity of potential candidate strains against the pathogen was evaluated through the dual culture method, followed by the identification of the dominant antagonistic strains by amplifying 16s ribosomal RNA for bacteria and ITS regions for fungi [17]. To prove the potential strains in the greenhouse, the control effect of the pathogen in potted R. lapponicum was also evaluated. The roots of R. lapponicum were inoculated first with mycelium suspension of pathogen Pci-1. After 1 week, the soil substrate of R. lapponicum pot was tested on a selective medium. A total of five spots were taken from each pot and placed on a PDA medium plate. After detection, seedlings carrying pathogen Pci-1 were used to check the biocontrol ability of potential candidate antagonists. Each treatment consisted of 5 pots of seedlings and each pot was irrigated with 100 ml candidate antagonists fermented suspension at a concentration of 1 × 10⁷ cfu/ml. The sterilized water was used as negative control and 40% Dimethomorph(SC) was the chemical (positive) control. Additionally, the soil substrate in the pot was regularly isolated and counted with a selective medium and the pathogen reduction rate was calculated accordingly.

2.7 Toxicity determination of chemical agents

2.8 Statistical analysis

The software SPSS 24.0 (SPSS Statistics for Windows, version 24.0; IBM Corp) was used for the analysis of variance. Means were subjected to Duncan's multiple range tests at p ≤ 0.05. All figures were processed and analyzed by using Adobe Illustrator CS6 (Adobe Systems Inc.) and GraphPad Prism (8.0.2).

3.1 Symptom description of root rot of Rhododendron lapponicum

At the early stage of infection, brown spots appeared on the surface of roots and stem and newly emerged leaves turned yellowish. Pathogens spread rapidly in the presence of moist soil. Infected leaves drooped and resulted in enlarged angles between leaves and stem. The root parts turned brown and black at a severe stage of symptoms. Plants started showing reduced growth and all leaves turned yellow, and finally, the whole plant weakened and dieback (Figure 1). Based on the symptoms mentioned above, it was assumed that the causal organism belongs to oomycetes. Furthermore, the pathogen was isolated from the infected root tissue by using the PDA and OA medium of Phytophthora. As a result, white and loose colonies were isolated from the infected roots and designated as a suspected isolate Pci-1 (Figure 2a).

3.2 Pathogenicity test for pathogen confirmation

After the mycelium suspension of isolate Pci-1 had been inoculated to R. lapponicum root for 45 days, the plants' growth stopped, leaves dehydrated, turned yellow, and drooped. In addition, the roots were also dehydrated and drought, turned brown and black, the whole plant withered, and finally died (Figure 1e–h). The symptoms were similar to root rot of R. lapponicum reported previously [7]. The morphological characteristics of the pathogen isolated from the infected plants were observed and the results were exactly the same as that of the first isolated Pci-1 (Figure 2b). Therefore, Koch's postulate confirmed the isolate Pci-1 as the pathogen of root rot on R. lapponicum.

3.3 Morphological characteristics and taxonomic status

3.3.2 Molecular identification of candidate Pci-1 strain

Genomic DNA of strain Pci-1 was used as a template, the amplified sequences of Btub-F1/Btub-R1 (1137 bp), ITS1/ITS4 (727 bp), and Yph1F/Yph2R (476 bp) were submitted to NCBI GenBank database with sequence numbers of MZ905503, MZ901370, and MZ905504, respectively. BLAST results showed that the strain Pci-1 had the closest genetic relationship with P. cinnamomi in GenBank database and the consistency was 100%. The phylogenetic tree based on β-tubulin gene of Pci-1 is displayed in Figure 3 and the phylogenetic trees based on the ITS and Ypt1 gene of Pci-1 are mentioned in Supporting Information: Figures S1 and S2. According to the above-mentioned results, strain Pci-1 was identified as Phytophthora cinnamomi Rands.

3.4 Host range

In this study, more than 60 species were tested (Supporting Information: Table S1), in addition to R. lapponicum, 14 species were infected by P. cinnamomi Pci-1, including Althaea rosea, Viburnum cylindricum, and Brassica napus. The diseased leaves were brown, expanded slowly, and decayed (Figure 4). The leaves resistant to disease had no obvious symptoms and the wounded ones were accrued from sclerenchyma.

3.5 Growth conditions

Isolate Pci-1 could grow significantly on all six media (Figure 5), but it grow most efficiently on the OA plate. The average diameter of the colony reached 69.30 mm after 5 days of culture, followed by V8, and minimal growth was noticed on PSA media. At 15–30°C, the colonies of Pci-1 were expanded on the OA medium, and the optimum temperature was 28°C. The colonies of Pci-1 were not observed on the OA plate when the mycelium was exposed to 40°C for 20 min, showing that the mycelium of Pci-1 could be completely inhibited with this range of temperature (data not shown).

Comparing different pH values, the results showed that Pci-1 could grow in the range of pH 4.0–10.0, and the largest colony diameter was observed on the OA plate with pH value of 7.0–8.0, indicating that the most suitable pH value for the growth of Pci-1 was 7.0–8.0. The growth on the OA plate with pH values of 4.0 and 10.0 resulted in smaller colonies indicating that exceedingly acidic or basic media were not conducive to the growth of Pci-1 on the OA plate.

3.6 Isolation and identification of candidate strains against Pci-1

The candidate antagonistic strains of Pci-1 were screened by dual culture method and identified as Paenibacillus polymyxa (MZ771306) (Figure S3) and Trichoderma asperellum (MZ771300) (Figure S4) by 16S rRNA and ITS, respectively. Among them, P. polymyxa P2-5 and Pci-1 were cultured in PDA medium at 28°C for 7 days (Figure 6a). The latter had no complete mycelial structure and was difficult to grow on a special medium. In addition, T. asperellum Tv-1 had obvious inhibitory and parasitic effects on Pci-1, which was limited to the initial colony before contact (Figure 6b). The pot experiment in the greenhouse suggested that the two antagonistic strains had a better control effect on Pci-1 than the fungicide. After 3 days of treatment, the pathogen load reduced significantly, and after 12 days, the pathogen declined to a considerable level of 28.38%–36.49% (Figure 6c).

3.7 Efficacy of fungicides against colony growth of Pci-1

The efficacy of the following six fungicides on Pci-1 was determined by the growth rate method (Table 2): 80% dimethomorph (water dispersible granule [WG], Shanxi Welch crop protection Co., Ltd.), 40% dimethomorph (suspension concentrate [SC], Jiangsu Jianpai Agrochemical Co., Ltd.), 30% flumorph (SC, Shenyang Kechuang Chemicals Co., Ltd.), 70% dimethomorph + cymoxanil (WG, Shandong Rongbang Chemical Co., Ltd.), 687.5 g/L fluopicolide + propamocarb (SC, Bayer Crop Science (China) Co., Ltd.), and 62.5 g/L mefenoxam + fludioxonil (flowable concentrate [FS], Shanghai Hulian Biopharmaceutical  Co., Ltd.). The selected fungicides had different degrees of inhibition on the colony growth of Pci-1. The order of EC₅₀ was as follows: fluopicolide + propamocarb > mefenoxam + fludioxonil > flumorph > dimethomorph > dimethomorph > dimethomorph + cymoxanil. Other four fungicides had higher antagonistic activity against isolate Pci-1, except for the two fungicides (Fluopicolide + propamocarb and mefenoxam + fludioxonil) (Table 2). The inhibitory effect of dimethomorph + cymoxanil on the isolate Pci-1 was significantly higher than others and the EC₅₀ value was 0.1894 a.i. µg/ml, EC₉₀ value was 0.3618 a.i. µg/ml.