Characterization and Control of Dendrobium officinale Bud Blight Disease

Dendrobium officinale is an important traditional Chinese medicine (TCM). A disease causing bud blight in D. officinale appeared in 2021 in Yueqing city, Zhejiang Province, China. In this paper, 127 isolates were obtained from 61 plants. The isolates were grouped into 13 groups based on collected areas and morphological observations. Four loci (ITS, LSU, tub2 and rpb2) of 13 representative isolates were sequenced and the isolates were identified by constructing phylogenetic trees with the multi-locus sequence analysis (MLSA) method. We found the disease to be associated with three strains: Ectophoma multirostrata, Alternaria arborescens and Stagonosporopsis pogostemonis, with isolates frequencies of 71.6%, 21.3% and 7.1%, respectively. All three strains are pathogenic to D. officinale. A. arborescens and S. pogostemonis isolated from D. officinale were reported for the first time. Iprodione (50%), 33.5% oxine-copper and Meitian (containing 75 g/L pydiflumetofen and 125 g/L difenoconazole) were chosen to control the dominant pathogen E. multirostrata, with an EC50 value of 2.10, 1.78 and 0.09 mg/L, respectively. All three fungicides exhibited an effective inhibition of activities to the growth of the dominant pathogen E. multirostrata on potato dextrose agar (PDA) plates, with Meitian showing the strongest inhibitory effect. We further found that Meitian can effectively control D. officinale bud blight disease in pot trial.


Introduction
Dendrobium officinale Kimura et Migo is a famous traditional Chinese medicine, which contains many bioactive components, such as polysaccharides, alkaloids, flavonoids and phenanthrene phenols [1,2]. D. officinale has been found to have functions related to immunity enhancement, the lowering of blood sugar, blood lipids and blood pressure, anti-oxidation, anti-tumor and cancer cell inhibition [3].
In nature, D. officinale grows in shady and humid rock crevices, and grows symbiotically with lichens, mosses, ferns and other plants [1]. It has been cropped in several provinces in China, such as Zhejiang, Guizhou and the Yunnan Province [4]. D. officinale is mostly cropped in greenhouses using pine scales, sawdust, wood and rocks as media, with drip and spray irrigation systems supplying water and fertilizer. Greenhouses maintain suitable temperature and humidity levels for the growth of D. officinale, as well as pathogens.
Stem diseases, such as stem dieback, can be caused by A. alternata [13] and Fusarium spp. [14,15], and stem rot can be caused by Lasiodiplodia theobromae, F. kyushuense and

Plant Materials, Pathogens Isolation and Purification
From 2021 to 2022, D. officinale plants (n = 61) with bud blight disease were collected from a greenhouse in Yueqing City (28.07 • N, 120.57 • E), Zhejiang Province, China. The incidence rate of the disease was assessed by visual observation of the presence or absence of symptomatic plants in the surveyed greenhouses. Pathogens were isolated according to the following method: the symptomatic plants were cut with a sterilized scalpel and rinsed with tap water for 15 min to remove dirt from the surface, then dried on tissue paper. Afterward, the symptomatic buds were cut into 4 mm 2 segments using a sterilized scalpel, superficially disinfected with 5% sodium hypochlorite solution (0.25% active ingredient of chlorine) for 1 min and 75% alcohol for 30 s, then washed with sterile distilled water 3 times, dried on sterile filter papers under aseptic conditions, and finally, the picked segments were placed onto PDA plates. The plates were subsequently incubated at 25 • C; in the dark, and the colonies were purified by the hyphal tip method [29] and then subcultured on the PDA and oatmeal agar (OA) media for morphological observation.

Pathogenicity Tests of Isolates
To test for pathogenicity, the fungal isolates were inoculated on the original host. The top three leaves were inoculated. The leaves were stabbed gently with sterile needles to cause tiny wounds, and the mycelial plugs (∅ = 6 mm) from 5-day-old cultures of the isolates were placed on the surfaces of the wounded leaves and wrapped with cling wrap. In contrast, the control plants received non-colonized agar plugs. All plants were covered with plastic bags to maintain moisture and then placed in a light incubator under conditions of 25 • C, 12 h dark/light. Each treatment had 3 replicates. All inoculated plants were observed for 20 days. Isolates causing necrosis over 4 mm 2 were considered pathogenetic. Fungal isolates which were re-isolated from inoculated plants were identified by rpb2 sequence data to fulfill Koch's postulates.

Morphological Observation
Purified isolates were grown on PDA and OA media at 25 • C in the dark for 7 days, after which morphological characteristics were observed and photographed. The microstructures of isolates were observed with a Nikon Eclipse Ni microscope with differential interference contrast (DIC) optics, equipped with a Nikon DS-Fi2 digital camera [30] and a jsz6 dissecting microscope. If necessary, near-UV light was used to promote the production of conidia [31].
PCR amplifications were performed in a total volume of 25 µL containing 13 µL 2×PCR buffer (Vazyme, Nanjing, China), 1 µL of each primer, and 1-10 ng genomic DNA. For LSU, ITS and tub2, the PCR amplification condition were: an initial denaturation for 3 min at 95 • C, followed by 35 cycles of 15 s at 95 • C, 15 s at 53 • C (for LSU and ITS) or 56 • C (for tub2), 1 min at 72 • C, with a final extension step for 5 min at 72 • C [41]. For rpb2, the PCR amplification condition were: an initial denaturation at 95 • C for 3 min, followed by 5 cycles of 15 s at 95 • C, 15 s at 60 • C and 1 min at 72 • C, then 5 cycles with a 58 • C annealing temperature and 30 cycles with a 54 • C annealing temperature, and a final extension step for 5 min at 72 • C [42]. PCR products were observed on 1% agarose gel. Sanger sequencing was conducted by Youkang Biotechnology Co., Ltd. (Hangzhou, Zhejiang Province, China). The accession numbers of all generated sequences in this study were further obtained from GenBank and listed in Table 1.

Phylogenetic Analysis
Phylogenetic constructions were made by maximum likelihood. All obtained sequences were compared in the Basic Local Alignment Search Tool (BLAST). Sequences of related species were downloaded from NCBI and listed in Tables S1-S3. Subsequent alignments for four individual loci (ITS, LSU, rpb2 and tub2) were generated with MAFFT v. 7 (https://mafft.cbrc.jp/alignment/server/, accessed on 10 April 2023) using default settings on a web server [43]. Gaps were considered to be missing data and alignments were manually adjusted for maximum alignment and sequence similarity. Sequences were cut to the same length using BioEdit v. 7.2.5. Concatenation and maximum likelihood analyses, including 1000 bootstrap replicates, were conducted using RAxML GUI v. 2.0.6. A general time-reversible (GTR) model was applied with a gamma-distributed rate variation. The resulting trees were viewed using MEGA 11 [33]. The mycelial growth rate method [44] was used to assess the sensitivity of the pathogen to the following fungicides: 50% iprodione (FMC, Los Gatos, CA, USA), 33.5% oxine-copper (Hong Yang Chemical Industry, Lvliang, China), 200 g/L Meitian (containing 75 g/L pydiflumetofen and 125 g/L difenoconazole, Syngenta, Nantong, China).
Fungicides were added to the PDA plate at final concentrations of 156.25, 31.25, 6.25, 1.25 and 0.25 mg/L for 50% iprodione; 33.5, 6.7, 1.34, 0.268 and 0.0536 mg/L for 33.5% oxine-copper; and 2, 0.4, 0.08, 0.016 and 0.0032 mg/L for Meitian (containing 75 g/L pydiflumetofen and 125 g/L difenoconazole). Mycelial plugs of the pathogen were placed at the center of the fungicide-amended PDA plates and incubated in the dark at 25°C for 7 d. Plugs placed on water-amended PDA plates served as the control. Each treatment had three replicates. The colony diameter was measured to evaluate the sensitivity of the pathogen to fungicide. Variance analysis and calculation of EC 50 values were performed using IBM SPSS Statistics v. 26 [44].

Control Test In Vivo
Healthy D. officinale plants were inoculated with pathogens using the same method in the pathogenicity tests of the isolates described above. When infective symptoms initially appeared, plants were removed from sampling bags for hours to dry. Of the Meitian (recommended minimum concentration in the field), 80 mg/L was evenly sprayed on the surface of the plants, and then whole pots of plants were put back in the incubator with a sampling bag to retain moisture. The control treatment was sprayed with an equal volume of sterile water. Each treatment had 6 replicates. The observation was carried out 20 days after inoculation.

Field Observation of Disease
In September 2021, D. officinale bud blight disease was found in Yueqing City, Zhejiang Province, China. It causes young buds to turn yellow and develop blight lesions which can spread to new leaves. Ultimately, the buds and 3 to 5 top leaves wither and the plants stop growing ( Figure 1). As far as we know, this study is reporting the disease, which we named Dendrobium officinale "bud blight" according to the symptoms, for the first time.
The disease mostly occurs from June to July, and September to October. During these periods, high temperatures, high humidity, and poor ventilation are conducive to the growth and reproduction of pathogens. The disease spreads rapidly in some greenhouses and the disease incidence was calculated to be over 50% using a random sample of 100 plants.

Grouping of Isolates and Phylogenetic Analysis
A total of 127 fungal isolates were isolated from 61 diseased plants and based on isolates' collected area and morphological traits, were grouped into 13 groups. Thirteen representative isolates were selected for further analysis. Each isolate came from different infected buds or leaves. Four loci (ITS, LSU, rpb2 and tub2) of the 13 representative isolates were sequenced and the accession numbers were listed in Table 1. Consistent with their morphological traits and ITS sequences, these fungi belong to three genera, encompassing Ectophoma, Alternaria and Stagonosporopsis, with frequencies of 71.6%, 21.3% and 7.1% (Table 2), respectively.
Pathogens 2023, 12, x FOR PEER REVIEW 5 of 15 and the disease incidence was calculated to be over 50% using a random sample of 100 plants.

Grouping of Isolates and Phylogenetic Analysis
A total of 127 fungal isolates were isolated from 61 diseased plants and based on isolates' collected area and morphological traits, were grouped into 13 groups. Thirteen representative isolates were selected for further analysis. Each isolate came from different infected buds or leaves. Four loci (ITS, LSU, rpb2 and tub2) of the 13 representative isolates were sequenced and the accession numbers were listed in Table 1. Consistent with their morphological traits and ITS sequences, these fungi belong to three genera, encompassing Ectophoma, Alternaria and Stagonosporopsis, with frequencies of 71.6%, 21.3% and 7.1% (Table 2), respectively. Further, maximum likelihood, phylogenetic trees were built using the MLSA method to identify pathogens at the species level. For Ectophoma strains, the final concatenated DNA sequence dataset comprised 150 isolates and consisted of 2205 characters, including alignment gaps (gene boundaries ITS: 649 bp, LSU: 680 bp, rpb2: 601 bp, tub2: 275 bp). Neocucurbitaria quercina (CBS 115095) served as an outgroup ( Figure S1). According to the  Further, maximum likelihood, phylogenetic trees were built using the MLSA method to identify pathogens at the species level. For Ectophoma strains, the final concatenated DNA sequence dataset comprised 150 isolates and consisted of 2205 characters, including alignment gaps (gene boundaries ITS: 649 bp, LSU: 680 bp, rpb2: 601 bp, tub2: 275 bp). Neocucurbitaria quercina (CBS 115095) served as an outgroup ( Figure S1). According to the phylogenetic tree (Figure 2), these 10 isolates were identified as Ectophoma multirostrata. The topology of the phylogenetic tree is consistent with N. Valenzuela-Lopez's research [33]. The full phylogenetic tree is in Figure S1. phylogenetic tree (Figure 2), these 10 isolates were identified as Ectophoma multirostrata. The topology of the phylogenetic tree is consistent with N. Valenzuela-Lopez's research [33]. The full phylogenetic tree is in Figure S1.   According to the phylogenetic trees, two isolates (isolate 7 and 9) were identified as Alternaria arborescens (Figure 3) and isolate 8 as Stagonosporopsis pogostemonis (Figure 4). The full phylogenetic trees of Alternaria arborescens and Stagonosporopsis pogostemonis are in Figures S2 and S3, respectively. According to the phylogenetic trees, two isolates (isolate 7 and 9) were identified as Alternaria arborescens (Figure 3) and isolate 8 as Stagonosporopsis pogostemonis (Figure 4). The full phylogenetic trees of Alternaria arborescens and Stagonosporopsis pogostemonis are in Figures S2 and S3, respectively.

Pathogenicity Assessment
One representative isolate from each strain was selected for the pathogenicity test. Blight symptoms were observed on buds and leaves 10 days after inoculation. The symptoms were consistent with those of the disease observed in the field. The fungal isolates were re-isolated from infected leaves, which fulfilled Koch's postulates. No symptoms appeared in the control group ( Figure 5). All three strains were pathogenetic to D. officinale.

Pathogenicity Assessment
One representative isolate from each strain was selected for the pathogenicity test. Blight symptoms were observed on buds and leaves 10 days after inoculation. The symptoms were consistent with those of the disease observed in the field. The fungal isolates were re-isolated from infected leaves, which fulfilled Koch's postulates. No symptoms appeared in the control group ( Figure 5). All three strains were pathogenetic to D. officinale.

Morphological Observation of Pathogens
After incubation of the dominant pathogen colonies, Ectophoma multirostrata, on an OA medium for 7 days, we observed that the mycelia were brown; the pycnidia were globose or subglobose, brown to dark brown, solitary or confluent; the conidiogenous cells were transparent; and the conidia were transparent, nearly olive-shaped, about 1 to 1.5 × 5 to 6.5 μm in size, with one or more rounded protrusions on the surface (Figure 6).

Pathogenicity Assessment
One representative isolate from each strain was selected for the pathogenicity te Blight symptoms were observed on buds and leaves 10 days after inoculation. The sym toms were consistent with those of the disease observed in the field. The fungal isola were re-isolated from infected leaves, which fulfilled Koch's postulates. No symptoms a peared in the control group ( Figure 5). All three strains were pathogenetic to D. officina

Morphological Observation of Pathogens
After incubation of the dominant pathogen colonies, Ectophoma multirostrata, on OA medium for 7 days, we observed that the mycelia were brown; the pycnidia we globose or subglobose, brown to dark brown, solitary or confluent; the conidiogeno cells were transparent; and the conidia were transparent, nearly olive-shaped, about 1 1.5 × 5 to 6.5 μm in size, with one or more rounded protrusions on the surface (Figure

Morphological Observation of Pathogens
After incubation of the dominant pathogen colonies, Ectophoma multirostrata, on an OA medium for 7 days, we observed that the mycelia were brown; the pycnidia were globose or subglobose, brown to dark brown, solitary or confluent; the conidiogenous cells were transparent; and the conidia were transparent, nearly olive-shaped, about 1 to 1.5 × 5 to 6.5 µm in size, with one or more rounded protrusions on the surface (Figure 6). Pathogens 2023, 12, x FOR PEER REVIEW Alternaria arborescens colonies were typically grayish to dark gray on PDA. Co were septate, slightly constricted near some septa, with few longitudinal septa, obc or ovate in shape (6.5 to 15.0 × 12.2 to 18 μm) (Figure 7). Stagonosporopsis pogostemonis colonies were white on PDA, but dark gray on OA nidiomata were solitary and covered with dense hyphae. Conidia were 1 to 1.5 × μm, olive-shaped, transparent, solitary and aseptate (Figure 8). Alternaria arborescens colonies were typically grayish to dark gray on PDA. Conidia were septate, slightly constricted near some septa, with few longitudinal septa, obclavate or ovate in shape (6.5 to 15.0 × 12.2 to 18 µm) (Figure 7). Alternaria arborescens colonies were typically grayish to dark gray on PDA. Co were septate, slightly constricted near some septa, with few longitudinal septa, obc or ovate in shape (6.5 to 15.0 × 12.2 to 18 μm) (Figure 7). Stagonosporopsis pogostemonis colonies were white on PDA, but dark gray on OA nidiomata were solitary and covered with dense hyphae. Conidia were 1 to 1.5 × μm, olive-shaped, transparent, solitary and aseptate (Figure 8). Stagonosporopsis pogostemonis colonies were white on PDA, but dark gray on OA. Conidiomata were solitary and covered with dense hyphae. Conidia were 1 to 1.5 × 5 to 7 µm, olive-shaped, transparent, solitary and aseptate (Figure 8).

Sensitivity Assessment of Pathogens to Fungicides In Vitro
As E. multirostrata was the dominant pathogen in all obtained isolates, it was used as the indicator pathogen in fungicide tests.

Sensitivity Assessment of Pathogens to Fungicides in Vitro
As E. multirostrata was the dominant pathogen in all obtained isolates, it was u the indicator pathogen in fungicide tests.
All three fungicides had an inhibitory effect on E. multirostrata, with Meitian h the strongest effect.

Sensitivity Assessment of Pathogens to Fungicides in Vitro
As E. multirostrata was the dominant pathogen in all obtained isolates, it was used as the indicator pathogen in fungicide tests.
All three fungicides had an inhibitory effect on E. multirostrata, with Meitian having the strongest effect.

Pot Trial of Meitian against Bud Blight Disease
Meitian, as the most effective inhibitor of E. multirostrata among the three fungicides, was selected for the pot trial. Twenty days after inoculation with E. multirostrata, no symptoms were present in plants sprayed with Meitian. The disease incidence in the experimental group was 0 ( Figure 11A), while blight symptoms appeared 100% in the control group ( Figure 11B). All three fungicides had an inhibitory effect on E. multirostrata, with Meitian having the strongest effect.

Pot Trial of Meitian against Bud Blight Disease
Meitian, as the most effective inhibitor of E. multirostrata among the three fungicides, was selected for the pot trial. Twenty days after inoculation with E. multirostrata, no symptoms were present in plants sprayed with Meitian. The disease incidence in the experimental group was 0 ( Figure 11A), while blight symptoms appeared 100% in the control group ( Figure 11B).

Pot Trial of Meitian against Bud Blight Disease
Meitian, as the most effective inhibitor of E. multirostrata among the three fungicides, was selected for the pot trial. Twenty days after inoculation with E. multirostrata, no symptoms were present in plants sprayed with Meitian. The disease incidence in the experimental group was 0 ( Figure 11A), while blight symptoms appeared 100% in the control group ( Figure 11B).

Discussion
As a traditional Chinese medicine, D. officinale is widely planted in multiple provinces in China and its value was over CNY 2.7 billion in 2020 [45]. However, D. officinale fungal diseases are becoming more serious with the increased scale of cultivation. Bud blight appeared recently with serious effects in some planting areas, but no pathogens have been reported yet. This study found that Ectophoma multirostrata, Alternaria arborescens and Stagonosporopsis could all cause D. officinale bud blight and E. multirostrata was the dominant pathogen.
Plant pathogen identification includes traditional and molecular methods. Traditional identification is based on morphological characteristics, growth characteristics, host range, and biochemical characteristics, etc. Molecular identification focuses on sequencing and comparison of conserved DNA sequences, such as ITS, but it is difficult to identify pathogens at the species level with individual steward genes [46]. Multi-locus sequence analysis (MLSA) is a method of aligning, cutting and joining two or more specific gene sequences to generate phylogenetic trees, and it has become a widely-accepted method in taxonomy due to its high resolution and convenience [47].
E. multirostrata has been reported to cause root rot disease in Celosia argentea, chickpea and green gram (Vigna radiata) [48]. E. multirostrata was originally classified in the genus Phoma, however, in an article published by N. Valenzuela-Lopez in 2018, it was classified into a new genus, Ectophoma, based on morphological structure and phylogeny [33]. In 2018, Xie et al. reported that Phoma multirostrata var. microspora can cause D. officinale leaf spot [10], with lesions appearing only on the back of the leaves and the isolate appearing white on the PDA plate. The E. multirostrata isolated in this study causes bud blight on D. officinale and appears brown on the PDA plate. Three loci which were used by Xie et al. have been sequenced: the sequence of the ITS, ACT (actin gene) and TEF (translation elongation factor) loci between the two strains share 99, 99 and 98% similarity, respectively. The difference between the two pathogens should be a topic for further research.
There are only a few existing reports on S. pogostemonis. It belongs to the genus Phoma and causes leaf spot and stem blight in Pogostemon cablin (Lamiaceae), but it has not been implicated in any disease of D. officinale. To the best of the author's knowledge, this study is the first report of S. pogostemonis isolated from D. officinale causing bud blight disease.
Mirghasempour et al. reported that five Fusarium species can cause D. officinale dieback disease, with the symptoms appearing as chlorotic, blighted and wilted leaves of the apical meristem, with the shoot tip showing dark brown necrosis, dieback and eventually shoot death [14]. The dieback and bud blight could be distinguished easily from each other by symptoms: dieback disease infects from the shoot tip, while bud blight disease infects from the bud and new leaves.
Meitian is a fungicide mixed with 75 g/L pydiflumetofen and 125 g/L difenoconazole, and has the same components as Miravis Duo, which is approved in the US. Pydiflumetofen is a succinate dehydrogenase inhibitor (SDHI) that disrupts energy production [60]. Difenoconazole is a sterol demethylation inhibitor that inhibits cell membrane ergosterol biosynthesis [28]. It has been reported that Meitian can effectively inhibit rose powdery mildew and cucumber powdery mildew in the field [61,62]. In current study, Meitian was proven to be effective in controlling D. officinale bud blight. Due to its low-toxicity and high efficiency, Meitian is a promising tool for controlling D. officinale diseases.

Conclusions
Bud blight in D. officinale was reported for the first time in the present study. The pathogens included E. multirostrata, A. arborescens and S. pogostemonis. A. arborescens and S. pogostemonis were isolated from D. officinale for the first time. Among these pathogens, E. multirostrata was the dominant pathogen, with isolates accounting for 71.6% of detected pathogens. Three fungicides were tested to control E. multirostrata in vitro, with Meitian displaying the best inhibition effect. Further, through pot trail assessment, we found that Meitian can effectively inhibit D. officinale bud blight.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pathogens12040621/s1, Table S1. GenBank accession numbers of strains used to identify Ectophoma strain in the phylogenetic analyses; Table S2. GenBank accession numbers used to identify Alternaria strains used in the phylogenetic analyses; Table S3. GenBank accession numbers used to identify Stagonosporopsis strains used in the phylogenetic analyses; Figure S1. Phylogenetic tree for Ectophoma Strains; Figure S2. Phylogenetic tree for Alternaria Strains; Figure S3. Phylogenetic tree for Stagonosporopsis Strain.   Table 1). The data presented in this study are openly available in NCBI. Publicly available datasets were analyzed in this study. These data can be found here: https://www.ncbi.nlm.nih.gov/ (accessed on 28 February 2023).

Conflicts of Interest:
The authors declare no conflict of interest.