Colletotrichum siamense Strain LVY 9 Causing Spot Anthracnose on Winterberry Holly in China

Winterberry holly (Ilex verticillata) is an economically valuable landscaping ornamental plant. Serious outbreaks have been reported, in its leaf tips curl upward, irregular black brown spots appear on leaves, and extensive defoliation is commonly observed. The incidence in Hangzhou was estimated at 50% and resulted in large economic losses for growers in 2018. Samples were collected from the main cultivation area in Zhejiang Province. In total, 11 fungal isolates were obtained from diseased leaves through a single-spore purification method, and isolate LVY 9 exhibited strong pathogenicity. Based on morphology and molecular phylogenetic analyses based on multilocus sequence typing of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), and chitin synthase (CHS-1) genes, we identified the pathogen as Colletotrichum siamense, causative agent of anthracnose of winterberry holly.


Introduction
Winterberry holly (Ilex verticillata) is a deciduous shrub that is especially valued for its masses of glossy, bright red berries in winter. Also known as Black Alder, its branches are often used for decoration during the holiday season [1]. Winterberry holly is widely cultivated in Europe, the United States, and other countries for its unique ornamental qualities and high adaptability. To date, there are more than 30 varieties cultivated and sold in Europe and America, such as "Oosterwijk", "Winter Red", "Berry heavy", and "A. Gray", among many others [2,3].
In China, in addition to their desirable floral qualities and broad marketability, especially in the Spring Festival [4]. The vivid red berries symbolize good luck and auspicious harvest. The cultivation area of winterberry holly has been rapidly expanding since 2006 in Zhejiang, Shandong, Fujian, Henan, Jilin, and Gansu provinces, as well as in other regions of China [3,5].
Fruit rot of deciduous holly (Ilex spp. L.) was recently reported as an emerging threat in nurseries in the mid-western and eastern USA [6]. Disease symptoms including early defoliation and withered or rotten fruit were observed throughout all of the fruit development and maturation period [7]. Previous work has indicated that multiple fungal pathogens such as Alternaria alternata, Diaporthe ilicicola, as well as other minor pathogens such as A. arborescens, Botryosphaeriaceae, Colletotrichum fioriniae, C.nymphaeae, D.eres, and Epicoccum nigrum can cause primary and/or secondary infections during the growing season through inoculation by wind, rain splash, and pollinators [8,9]. To date, there have been very few reports describing diseases of winterberry holly, so the relevant disease resistance mechanisms remain unclear.
In 2018, growers reported the occurrence of a new anthracnose disease of winterberry holly in Zhejiang province, China, with primary symptoms including black or brown leaves, irregular leaf spots, and sparse white mycelium. The incidence of diseased leaves In 2018, growers reported the occurrence of a new anthracnose disease of winterberry holly in Zhejiang province, China, with primary symptoms including black or brown leaves, irregular leaf spots, and sparse white mycelium. The incidence of diseased leaves ranged from 10% to 50%, severely affecting the aesthetic quality. It is of great economic significance determine the pathogenic agent underlying this disease so that a timely and effective management strategy can be deployed. Here, in this work, we isolate the causative agent of this new disease, and identify it as Colletotrichum siamense.

Sample Collection and Pathogen Isolation
The symptomatic samples were collected from Zhejiang province in 2018 ( Figure 1). Diseased leave tissues were surface-sterilized with 75% ethanol for 30 s, and then 0.1% mercuric chloride solution for 3 min, rinsed five times by sterile distilled water, and air dried; the axenic tissues were subsequently cut into about 25 mm 2 pieces and plated on potato dextrose agar (PDA) medium containing 0.2 mg/mL −1 ampicillin sodium. The plates were incubated at 28 °C in the dark for 7-14 days. Pure mycelia were obtained by hyphae tip separation and stored in PDA slants covered by paroline at 4 °C [10,11].

Pathogenicity Test
Two inoculation methods were used for the pathogenicity assay. The vitro leaf method was described by Lin and Dissanayake [8,9,12]. One-month-old healthy leaves were collected from potted plants (A.Gray), surface-sterilized with 75% ethanol and immersed in sterile water-Tween 80 solutions (0.3% v/v) 3 times before inoculation. About 5 mm-diameter mycelial plugs were picked up from the culture medium and placed on left and right sides of the same leaf, then those leaves were incubated in a 10 cm 2 petri dish containing a wet sterile filter paper, inoculated with sterile PDA as controls. There were 3 replicates of each isolate and the experiment was repeated three times. The dishes were placed in a growth chamber under artificial light (12/12 h light/dark) at 28 °C for 7 days. The second inoculation technique was described by Weir [13]: 1 × 10 6 conidia/mL of the spore suspensions were prepared in sterile distilled water. Leaves were wounded evenly by the sterilized needle, and then the front and back of the leaves of the biennial plants were sprayed with the suspensions. Sterile distilled water was used as control. Then, all plants were maintained in a greenhouse under 100% relative humidity at temperatures between 30 °C (day) and 28 °C (night) with natural photoperiods. The pathogen was re-

Pathogenicity Test
Two inoculation methods were used for the pathogenicity assay. The vitro leaf method was described by Lin and Dissanayake [8,9,12]. One-month-old healthy leaves were collected from potted plants (A.Gray), surface-sterilized with 75% ethanol and immersed in sterile water-Tween 80 solutions (0.3% v/v) 3 times before inoculation. About 5 mmdiameter mycelial plugs were picked up from the culture medium and placed on left and right sides of the same leaf, then those leaves were incubated in a 10 cm 2 petri dish containing a wet sterile filter paper, inoculated with sterile PDA as controls. There were 3 replicates of each isolate and the experiment was repeated three times. The dishes were placed in a growth chamber under artificial light (12/12 h light/dark) at 28 • C for 7 days. The second inoculation technique was described by Weir [13]: 1 × 10 6 conidia/mL of the spore suspensions were prepared in sterile distilled water. Leaves were wounded evenly by the sterilized needle, and then the front and back of the leaves of the biennial plants were sprayed with the suspensions. Sterile distilled water was used as control. Then, all plants were maintained in a greenhouse under 100% relative humidity at temperatures between 30 • C (day) and 28 • C (night) with natural photoperiods. The pathogen was re-isolated from leaf spots, and its identity confirmed by morphological characteristics, which is consistent with the Koch's postulates.

Morphological Characteristics
Mycelia discs (7 mm in diameter) from 7-day-old PDA cultures were sub-cultured on PDA medium in incubator, at 28 • C. The mycelium growth was measured every day for Microorganisms 2023, 11, 976 3 of 11 7 consecutive days, and the colony features (size and color) were recorded after 7 days [14]. The mycelia were removed from the surface of the plate to enhance the sporulation under a mixture of fluorescent white and UV light with 12 h dark at 20 • C for 15-20 days [13,15,16]. The conidia were examined visually and microscopically for morphological features, the mycelia (7 mm diameter) with conidia were observed in SEM (Hitachi Model SU-8010) [17][18][19].
Appressorium was produced using a slide culture technique [13]. A mycelium colony on PDA with 1 cm2 was placed in an empty petri dish (9 cm) and immediately covered with a sterile cover slip, then transferred into an empty square petri dish (10 cm) containing sterile water. After 14 days, the cover slip was removed, and a drop of lactic acid was added on a glass slide [13,14].  Table 1.

Fungal Isolates Phylogenetic Analysis
The obtained sequences were edited by BioEdit 7.1.3.0 [24] and multiple alignments were generated with MAFFT 7.273 [25]. To construct the phylogenetic tree, available data for species of Colletotrichum were downloaded from the GenBank database (Table A1). Maximum likelihood (ML) analyses were performed using IQ-TREE [26,27] with the concatenation of the ACT, CAL, CHS-1, GAPDH, and ITS regions. The best evolutionary model was selected through Model Finder [28], as follows: TIM2 + F + R4 model for ACT, TIM + F + R3 for CAL, TIM + F + R3 for CHS-1, HKY + F + R2 for GAPDH and TIM2 + F + R3 for ITS. Confidence of the branch points was evaluated using 5000 bootstrap replicates. The ML values equal to or above 75% values were shown on a tree for significantly supported nodes. Bayesian Inference (BI) was applied to generate a phylogeny tree by MrBayes ver.3.2.6 [29]. GTR + F + I + G4 model was used to the analyses of the gene of ACT, CAL, CHS1, ITS and the HKY + F + G4 model for GAPDH following Ronquist and Huelsenbeck [29] for BI analyses with 200,000 replicates and the phylogenetic tree was sampled every 100 generations. The first 500 trees were removed for the burn-in phase. The consensus of the remaining trees was constructed with resulting node frequencies that were treated as Bayesian posterior probabilities (PP). BI posterior probability (BI-PP) values equal to or above 0.95 were determined to be significant. The tree was rooted with the outgroup, Monilochaetes infuscans.

The Strain LVY 9 Was Pathogen of Anthracnose on Winterberry Holly through Koch's Postulates
We identified and isolated eleven isolates (LVY 1-11) from symptomatic leaves of Ilexverticillata (Hangzhou city, Zhejiang province, China) based on morphological features and confirmation by Sanger sequencing. These isolates belonged to genera Colletotrichum, Alternaria, Botryosphaeria, Leptosphaeria, Cercospora, and Emericella, and subsequent pathogenicity tests indicated that only inoculation with Colletotrichum (isolate LVY 9) resulted in symptom formation on leaves in vitro. These symptoms included brown necrotic lesions with dense whitish-grey aerial mycelia, and a few bright orange conidial masses near the point of inoculation at 7 days post infection (dpi) (Figure 2a,b). The mycelia are grayish brown with white edge and reserve is turquoise after strain LVY 9 on PDA for 7 days. Colonies were round, swell, neat edge, with cottony radial growth of white. In particular, large clusters of bright orange conidia were produced on PDA for 30 d. These characteristics are very similar to those of Colletotrichum gloeosporioides complex. Following spray inoculations, similar symptoms appeared at 30 dpi in winterberry nursery fields ( Figure 2c). In diseased plants, leaf tips of inoculated plants exhibited an obvious curly phenotype (Figure 2d). In addition, we observed irregular gray black spots on the leaves. We were able to successfully re-isolate the Colletotrichum isolates from these diseased plants, thereby fulfilling Koch's postulates.
To characterize the microscopic features of the pathogen, in addition to its symptoms and colony morphology, we selected a representative isolate for morphological characterization by SEM and light microscopy. On PDA medium, isolate LVY 9 colonies first appeared white, then became gray to dark grey, with an average growth rate of approximately 10.72 mm/day. Bright orange conidia and conidiophores were produced under continuous UV + fluorescent white lights with a 12:12 h light: dark cycle (Figure 2g). Scanning electron microscopy revealed that conidial dimensions were 7.8-12.7 × 2. 6-3.6 µm, and conidia were smooth-walled, hyaline, and cylindrical, with obtuse to slightly rounded ends (Figure 3a-c). Under light microscopy, appressoria (5.8-10.7 × 3.9-7.7 µm) were observed to form mostly from mycelium on slide cultures, and appeared brown to dark brown, ovoid, clavate, and slightly irregular in shape (Figure 3d-h). Morphological features of the isolate LVY 9 were highly similar to those of species belonging to the Colletotrichum gloeosporioides complex [13,[30][31][32].

Strain LVY 9 Was Identified as Colletotrichum siamense by Phylogenetic Analyses
For rigorous molecular identification, we used partial sequences from ACT (GenBank number: OQ652092), CAL (GenBank number: OQ652091), GAPDH (GenBank number: OQ652089), CHS-1 (GenBank number: OQ652090), and ITS (GenBank number: OQ651128) from isolate LVY9 for species level identification of the pathogen (see Table 1 for primers and source studies). Maximum likelihood-based phylogenetic reconstruction of these concatenated sequences (2370 nucleotides) including ACT (1-311 bp), CAL (312-1119 bp), CHS-1 (1120-1420 bp), GADPH (1421-1736 bp), and ITS (1737-2370 bp) was congruent with that of a Bayesian inference (BI) tree for the five concatenated loci. Relationships among almost all of the reference isolates could be clearly distinguished at the species level. Furthermore, phylogenetic analysis indicated that isolate LVY 9 clustered with C. siamense CBS 130,420, C. siamense ICMP 18,587, and C. siamense CBS 125,378, thus forming a distinct clade, which was highly supported in both ML-BS (100%) and BI-PP (1.0) models (Figure 4). Based on these findings, we concluded that the LVY9 isolate was a strain of C. siamense, the previously reported causative agent of anthracnose in winterberry holly. To characterize the microscopic features of the pathogen, in addition to its symptoms and colony morphology, we selected a representative isolate for morphological characterization by SEM and light microscopy. On PDA medium, isolate LVY 9 colonies first appeared white, then became gray to dark grey, with an average growth rate of approximately 10.72 mm/day. Bright orange conidia and conidiophores were produced under continuous UV + fluorescent white lights with a 12:12 h light : dark cycle (Figure 2g). Scanning electron microscopy revealed that conidial dimensions were 7.8-12.7 × 2. 6-3.6 µm, and conidia were smooth-walled, hyaline, and cylindrical, with obtuse to slightly rounded ends (Figure 3a-c). Under light microscopy, appressoria (5.8-10.7 × 3.9-7.7 µm) were observed to form mostly from mycelium on slide cultures, and appeared brown to dark brown, ovoid, clavate, and slightly irregular in shape (Figure 3d-h). Morphological features of the isolate LVY 9 were highly similar to those of species belonging to the Colletotrichum gloeosporioides complex [13,[30][31][32].

Figure 2.
Symptoms of winterberry holly leaves after incubation with isolate LVY 9. In vitro inoculation, brown necrotic lesions with bright orange conidial masses appear (b) compared with control (a) after 7 days. The arrow points to orange conidia (b). In spray inoculation, grayish brown spots appear on tips of leaves with obvious curly phenotype compared with control (c) after 30 days. The arrow points to irregular spots (d). The mycelia are grayish brown with white edge (e) and reserve is turquoise (d) after strain LVY 9 on PDA for 7 d. Orange masses of conidia released from pycnidia after strain LVY 9 under a mixture of fluorescent white and UV light on PDA 20 d (g,h). The arrow points to orange conidia (g). Strain LVY 9 resembles Colletotrichum gloeosporioides by SEM and light microscopy. . Conidia and appressorium characteristics of isolate LVY 9 on PDA. Conidia are smoothwalled, hyaline, and cylindrical, with obtuse to slightly rounded ends (a-c). Bars = 10, 2, 1 µm, respectively; appressoria appear brown to dark brown, ovoid, clavate, and slightly irregular in shape(d-g). Bars = 20, 10, 10, 5 µm, respectively.
In the Colletotrichum gloeosporioides complex, C. gloeosporioides, C. siamense, and C. fructicola exhibit similar morphological characteristics including conidial size, shape, and appressoria formation. C. siamense is genetically close to C. fructicola, but C. fructicola has slightly longer and narrower cylindrical or subcylindrical conidia that have irregularly shaped, crenate, brown to dark brown appressoria, and branched hyphae [36,45]. However, the conidia of C. siamense are fusiform with obtuse to slightly rounded ends, occasionally oblong and ovoid. Previous reports also confirm that this species exhibits regular-to slightly irregular-shaped appressoria [14]. In contrast, C. gloeosporioides conidia appear cylindrical, although slightly tapered with obtuse or slightly rounded to oblong ends. The C. gloeosporioides appressoria are circular to slightly irregular, thus differing from those of C. siamense [14,46]. In this study, the shapes of the conidia and appressoria of isolates matched those of C. siamense, described by Prihastuti [14]. Moreover, the size of appressoria was similar to that of C. siamense, although the conidia described by Sharma [36,47] were smaller. Mycelial growth rates of isolates obtained in this study were also slower than those of the strain of C. siamense described by Yaowen [48]. We speculated that these morphological differences may be due (at least in part) to responses to the environment, growth media, and host.
Since morphological identification is important but not definitive for Colletotrichum, sequence analysis and infection behavior are also used to discriminate down to species level. As mentioned in previous studies, species in the C. gleosporioide species complex are genetically distinct from those in the C. boninense complex, but have highly similar micro-morphologiesm [13,15,16,49]. Initial sequence analyses relying on ITS were unable to satisfactorily distinguish among Colletotrichum species due to their high level of evolutionary conservation. In this work, we therefore used several genes to identify the LVY9 strain that were previously reported to successfully resolve Colletotrichum species [18,50]. Notably, ITS can separate C. gloeosporioides from all other Colletotrichum species, but cannot reliably separate C. siamense from C. alienum, C. fructicola, or C. tropicale. These species are best distinguished using CAL or TUB2 [13]. The causative agent of leaf spots on Sterculia nobilis in China was identified as C. siamense using a combination of ITS, ACT, GAPDH, CAL, CHS-1, and TUB2 genes [48]. In this study, we used a combinations of five genes (ACT, CAL, CHS-1, GAPDH and ITS) to identify the isolates obtained from winterberry holly, thus providing strong molecular evidence for the identification of the isolates as C. siamense.
C. siamense was originally described as a pathogen of coffee berries in Thailand [14]. This species has since been confirmed to infect more than 60 plant species worldwide [13,15,16]. This paper presents the first report describing C. siamense as the causal agent of anthracnose of winterberry holly in Zhejiang, China. As winterberry holly is commonly used as a bonsai or cut flower, the disease directly reduces its ornamental value. In addition, the occurrence of disease may harm the introduction and cultivation of winterberry holly. This identification of the disease-causing species facilitates the establishment of control measures, not only for winterberry holly, but for all of the known hosts of this pathogen. This study also provides a basis for future studies of the molecular mechanisms of this pathogenic interaction, identification of disease-resistant varieties, and creation of stable resistant materials.

Conclusions
According to Koch's rule, The strain LVY9 was elected as the pathogen causing anthracnose of Winterberry Holly in Zhejiang Province, China. The strain LVY9 was identified as the C. gloeosporioides complex by observing the morphology features of mycelia, conidia and appressorium. The strain LVY9 was identified as C. siamense by further analysis of the phylogenetic tree that combined Actin, Calmodulin, Glyceraldehyde-3-Phosphate dehydrogenase, Chitin synthase and Internal transcribed spacer genes. So, we concluded that C. siamense was causative agent of anthracnose in winterberry holly.

Acknowledgments:
We are grateful for technical support and practical experimental shared by Youxiang Yu which is a staff of HangZhou Runtu Horticulture Technology Co., Ltd. In addition, we thank Seyed Ali Mirghasempour khoshkroudi and Yunfang He for proofreading the manuscript.

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