First Report of Colletotrichum aenigma causing Anthracnose on Pecan in China.

Pecan (Carya illinoinensis) is an important economic forest crops widely cultivated in China. From June to September in both 2021 and 2022, severe leaf disease resembling anthracnose was observed in 6.6-ha pecan orchard in Jintan (31°42'23.84″ N, 119°21'22.90″ E), Jiangsu Province. The disease severity was about 15 to 25% with 5 to 12% incidence on 100 surveyed trees of the orchard in 2022. Symptoms initially appeared as small gray-bark sunken lesions, which gradually developed to big sunken lesions with brown edges and irregular-shaped. Small fragments (4 × 4 mm) from the necrotic borders of infected leaves were surfaced sterilized, plated on potato dextrose agar (PDA) and then incubated in darkness at 25°C for 3 days. Pure cultures were obtained by monosporic isolation. Twenty-one isolates with similar characteristics were obtained from the infected leaves (isolation frequency about 90%). The upper side of colonies on the PDA plates was milky, and the reverse side was pale yellow at the center and pale white at the margin. After 10 days of growth on the PDA medium, these isolates produced spores separately. . Through electron microscopic observation, conidia were smooth walled, hyaline, aseptate, guttulate, cylindrical with rounded ends with 15 to 20.5 × 5.3 to 6.7 µm (mean 18.5 × 5.8 µm, n = 50) in size. These morphological characteristics were similar to those of the species of Colletotrichumspp (Weir et al. 2012, Fu et al. 2019). To further identify the isolates, the regions of internal transcribed spacer (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHSI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin 2 (TUB2) loci of the three representative isolates (JSJT-1, JSJT-2, and JSJT-3) were amplified and sequenced with the primer pairs ITS-1F/ITS-4, ACT-512F/ACT-783R, CL1/CL2A, CHS-79F/CHS-345R, GDF/GDR and T1/T2 primers, respectively (Weir et al. 2012). Sequences of them were deposited in GenBank under nos. OR214960 to OR214962 (ITS), OR228543 to OR228545 (ACT)，OR228546 to OR228548 (CAL), OR228549 to OR228551 (CHSI), OR228552 to OR228554 (GAPDH), and OR228555 to OR228557 (TUB2). Multilocus phylogenetic analysis revealed that the three isolates and C. aenigma were clustered in the same clade. Based on the results of morphological and molecular analysis, these isolates were identified as C. aenigma. The pathogenicity of three isolates was tested on leaves of pecan seedlings. Suspensions of conidia were obtained by scraping the surface of a 10-day-old sporulated petri dish PDA cultures into sterile water. Suspensions were adjusted to a density of 2 × 106 conidia/ml with a hemocytometer.The conidial suspension of each isolate was sprayed evenly on the surface of leaves from three healthy pecan seedlings. Sterilized distilled water was used for negative controls. The pathogenicity experiment was repeated three times. Finally, all inoculated plants were kept in a light-incubator at 28°C under 100% relative humidity and 12 h photoperiod. Two weeks after inoculation, the inoculated plants developed symptoms similar to those of the original diseased plants, while controls remained asymptomatic. C. aenigma were re-isolated from from inoculated leaves. C. aenigma has been reported as the causal agent of anthracnose on several economically important plants, such as grape ( Kim et al. 2021), tree peonies (Wang et al.2023), chili (Diao et al. 2017), and pear (Fu et al. 2019), but this is the first report of C. aenigma causing anthracnose on pecan in China. Identification of C. aenigma as a pathogen of pecan is important for implementing control management strategies for pecan disease. References: Diao, Y. Z., et al. 2017. Persoonia. 38:20. Fu, M., et al. 2019. Persoonia. 42:1. Kim, J. S., et al. 2021. Plant Dis. 105:2729. Weir, B. S., et al. 2012. Stud. Mycol.. 73:115. Wang, Y. L., et al. 2023. Plant Dis. 107(4):1242. The author(s) declare no conflict of interest. Keywords: Colletotrichum aenigma, Anthracnose, Carya illinoinensis, Pathogenicity.

First Report of Anthracnose on Ilex cornuta caused by Colletotrichum aenigma in China.

Ilex cornuta (Aquifoliaceae) is a dark green evergreen shrub with glossy leaves that is widely distributed in China and East Asia and used as an ornamental and medicinal plant. In March 2022, typical symptoms of anthracnose were observed on I. cornuta leaves (with approximately 30% of leaves affected) in Jiangxi Academy of Forestry, Nanchang city, Jiangxi Province, China. The early symptoms were light brown spots on the edge or tip of the leaves. The spots gradually expanded to ovoid-shaped lesions and eventually become necrotic, dry, and gray with a dark brown margins. To isolate the pathogen, ten symptomatic leaves were randomly collected, the edges between diseased area and healthy area were cut into small pieces (4×4 mm), surface sterilized by dipping in 70% ethanol for 30 s and 1% sodium hypochlorite (NaClO) for 30 s, and then washed three times with sterile distilled water. Leaf pieces were then placed on potato dextrose agar (PDA) plates at 28℃ in the dark. Subsequently, six isolates were obtained using the single-spore method, five of them were similar in morphological characteristics. Colonies grown on PDA for 7 days were 75-85 mm in diameter, and were cottony, dense, and pale white on the surface and white to grayish-green on the reverse side. Conidia were single-celled, transparent and subcylindrical to clavate. The contents of the conidia were granular and 15.63-20.63 × 5.63-7.50 µm in size (=17.78 ± 1.41× 6.50 ± 0.55 µm, n = 40). The species was also identified by analysis of the internal transcribed spacer (ITS) region, actin (ACT), glyceradehyde-3-phosphate dehydrogenase (GAPDH) and ß-tubulin (TUB2), calmodulin (CAL), glutamine gynthetase (GS), DNA Lyase (APN2), intergenic spacer and partial mating type (ApMat) genes using ITS4/ITS5, ACT-512F/ACT-783R, GDF/GDR, T1/Bt2b, CL1C/CL2C, GSF/GSR, ColDL-F3/CgDL-R1 and CgDL-F6/CgMAT1F2 primers, respectively (Weir, et al. 2012; Maharachchikumbura, et al. 2014; Khodadadi, et al. 2020). The sequences were deposited in GenBank (Accession Nos. OQ600619, OQ603370, OQ603373, OQ603379, OQ974177, OQ974176, OQ974178, and OQ974175). BLASTN analysis in GenBank showed that these genes exhibited 100% similarity to the sequences of Colletotrichum aenigma (MT476812, MN525817, MN525878, MN525904, MN525836, KX620296, and MN338281) and 99% similarity to the sequence of Colletotrichum sp. strain (MT071110). Concatenated sequences of these eight genes and Colletotrichum species sequences from GenBank were then used to construct a phylogenetic tree by using the maximum likelihood method in IQtree V1.5.6. Isolate JFRL 03-1005 was grouped into a clade with C. aenigma with a high bootstrap value. Thus, the isolate was identified as C. aenigma based on morphological and molecular data. To verify Koch's postulates, pathogenicity was tested on 2-year-old healthy potted plants of I. cornuta. Ten disinfected leaves were wounded with a sterile scalpel, and then inoculated with 10 µl of conidial suspension (1 × 106 conidia/ml) from isolate JFRL 03-1005. The control leaves were inoculated with 10 µl of sterile water. Then, the potted plants were incubated at 28°C with a 12 h photoperiod and 80% humidity. After 10 days, distinct spots appeared on all inoculated leaves, whereas control leaves remained asymptotic. C. aenigma was reisolated from the spots and identified by sequencing the ITS, ACT, GAPDH, TUB2, CAL, GS, APN2 and ApMat genes. Previous studies reported that C. aenigma can caused anthracnose on the leaves of various cash crops in China, such as apple, tree peonies, mulberry, and walnut (Wang et al. 2020; Zhang et al. 2021; Wang et al. 2022; Zhu et al. 2022). To the best of our knowledge, this is the first report of C. aenigma causing anthracnose on I. cornuta in China, this report further confirmed that C. aenigma has a wide range of hosts in nature. the anthracnose on I. cornuta caused by C. aenigma has seriously affected its ornamental value. Therefore, more attention should be paid to this disease and appropriate control strategies should be formulated.

First Report of Colletotrichum aenigma Causing Anthracnose of tree peony in China.

Tree peonies (Paeonia suffruticosa Andr. and hybrids) are well-known ornamental and medicinal plants cultivated in temperate and subtropical regions around the world. From June to September 2021, severe leaf spot disease was observed on 3 tree peony cultivars ('Luoyanghong', 'Moyushenghui', 'Roufurong') in Xinxiang (35º29´N, 113º95´E) and Luoyang (34º64´N, 112º49´E), Henan Province, China. Leaf spot incidence was as high as 28% ('Luoyanghong'), 45% ('Moyushenghui') and 67% ('Roufurong'), respectively. Symptoms appeared initially as small purple spots less than 1 mm in diameter in the middle and upper parts of the leaves, and then evolved to coalescent lesions, causing brown scorch ultimately. From each cultivar, 5 diseased leaves were collected. Leaflet tissues (3-4 mm2) cut from spot margins were surface sterilized in 75% alcohol for 45 s, washed 5 times with sterile distilled water, and then cultivated on potato dextrose agar (PDA) medium at 28 °C in the dark. Eleven isolates were obtained, and colonies grown from single conidia on PDA were 80-85 mm in diameter after 10 d, with scattered small, dark-based spikes on the surface of the colonies. The aerial mycelium was cottony, dense, and dark gray near the center on the reverse side. Conidia were cylindrical to clavate, with rounded apex and rounded base, and the conidia contents were granular, 8.44-14.06×3.60-4.31 µm (mean=11.28×3.69 µm, n=40). Appressoria were mostly subglobose or with a few broad lobes, pale to medium brown, 3.36-6.72×3.35-5.60 µm (mean=5.02×4.55 µm, n=20). Based on the culture representation and conidial morphology, the isolates were characterized as Colletotrichum gloeosporioides species complex (Weir et al. 2012; Fu et al. 2019). To further identity the species, the actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the ribosomal internal transcribed spacers (ITS) loci of isolates PSW0002, PSW0008 and PSW0009 were amplified using ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-345R, GDF/GDR, and ITS1/ITS4, primers (Weir et al. 2012; Schena et al; 2014; Kim et al. 2021; Li et al. 2021). Fifteen sequences were deposited in GenBank (ACT for OP225605, OP225606, and OP225607, CAL for OP225608, OP225609 and OP225610, CHS for OP225611, OP225612 and OP225613, GAPDH for ON321897, OP225614, and OP225615, and ITS for ON323473, OP214349 and OP214350 ), which showed 100% sequence similarity to Colletotrichum aenigma (JX009443 and JX009519 for ACT, JX009683 and JX009684 for CAL, JX009774 and JX009903 for CHS-1, JX010244 and JX009913 for GAPDH, JX010243 and JX010148 for ITS). Three isolates clustered with C. aenigma (ex-holotype culture ICMP 18608) in the multi-locus phylogenetic tree with a bootstrap value of 100%. To achieve Koch's postulates, pathogenicity was tested on 5-year-old healthy potted plants ('Luoyanghong'). Thirty leaves were inoculated with 10 µL conidial suspension (isolate PSW0002, 1×106 conidia/mL) and the controls were inoculated with sterile water. Plants were placed in a greenhouse at 28°C under conditions with 12 h photoperiod and 90% relative humidity. After 5 to 10 days, distinct spots were observed on the inoculated leaves, while the control leaves showed no symptoms. C. aenigma was reisolated from all inoculated leaves, but not from the control. C. aenigma has been reported to cause anthracnose on Pyrus pyrifolia (Weir et al. 2012), Camellia sasanqua (Chen et al. 2019), Juglans regia (Wang et al. 2020), Paeonia ostii (Ren et al. 2020), and Capsicum annuum (Sharma et al. 2022). A previous study reported C. gloeosporioides as a pathogen of anthracnose in tree peonies in China (Xuan et al. 2017), the typical symptoms were big necrotic lesions (5-10 mm diam) on leaves，which were significantly different from those caused by C. aenigma. To our knowledge, this is the first report of C. aenigma causing anthracnose in tree peonies in China. This finding may help to take effective control of anthracnose in tree peonies.

First Report of Colletotrichum aenigma Causing Anthracnose on Mulberry Leaves in China.

Mulberry (Morus alba L.) has been grown worldwide as a crop for silkworm rearing for over five thousand years (Jiao et al. 2020). In July 2021, a leaf spot disease was observed on mulberry leaves in Wuhan city (114°33'E, 30°48'N), Hubei province, China, with approximately 40% of leaves (about 300 trees) affected. Early symptoms were light brown, with small lesions subsequently expanding to larger sometimes irregular dark brown or black spots surrounded by yellow-brown margins, with easily perforated necrotic lesions. Leaf tissues (5 mm×5 mm) were excised from the border between diseased and healthy tissues, surface sterilized with 75% ethanol solution for 30 s and 2.5% sodium hypochlorite for 2 min, washed thrice in sterile distilled water, and then placed on potato dextrose agar (PDA), and incubated at 25°C in darkness. Four isolates (C1, C9, CHS2, and CHS6) were subcultured using the single-spore method. On PDA, colonies were cottony, pale white from above, and white to grayish-green on the reverse side. Conidia were aseptate, hyaline, subcylindrical with broadly rounded ends, 8.4 to 18.3×4.1 to 7.7 µm (mean = 13.9×5.5 µm, n = 30). Appressoria were typically elliptic or irregular with a few lobes, dark brown, 5.9 to 9.6×4.2 to 8.1 µm (mean = 7.9 ×5.7 µm, n = 30). The morphological characteristics of the isolates matched the descriptions of Colletotrichum gloeosporioides species complex (Weir et al. 2012). The isolates were further identified by analysis of the ribosomal internal transcribed spacers (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), actin (ACT), chitin synthase (CHS-1), glutamine synthetase (GS), and ß-tubulin 2 (TUB2) genes, amplified respectively with ITS1/ITS4, GDF/GDR, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-345R, GSF1/GSR, and Bt2a/Bt2b (Glass and Donaldson 1995; Weir et al. 2012; White et al. 1990). The sequences were deposited in GenBank (ON492187-ON492214). Concatenated sequences of the seven genes in addition to Colletotrichum species sequences from GenBank were used to conduct a phylogenetic analysis using Maximum-Likelihood (ML) method in MEGA7. The four isolates were grouped into a clade with Colletotrichum aenigma supported by a high bootstrap value (89%), and hence, they were identified as C. aenigma based on the morphological and molecular analyses. To confirm Koch's postulates, wounded leaves of six healthy 2-month-old seedlings made by a sterile needle were inoculated with each isolate by spraying 10 ml of conidial suspensions (105 conidia/ml) on each plant, and the control plants were treated with sterile distilled water. All the treated plants were kept in a plastic box containing sterile water and incubated at 28°C in a 12 h/12 h light/dark cycle. The test was performed three times. After 7 days, typical anthracnose lesions appeared on all inoculated leaves, whereas control plants remained asymptotic. Furthermore, C. aenigma was only reisolated from the symptomatic leaves. Previous studies reported five Colletotrichum species (C. morifolium, C. fioriniae, C. brevisporum, C. karstii, and C. kahawae subsp. ciggaro) to cause this disease on mulberry in China (Tian, 1981; Xue et al. 2019). To our knowledge, this is the first report of C. aenigma causing anthracnose on mulberry in China. The finding will facilitate epidemiological studies and the development of effective control strategies for the disease.

Fluorescent Labeling of Peroxisome and Nuclear in Colletotrichum aenigma.

Anthracnose is one of the most widespread and destructive diseases in grapes. Grape anthracnose can be caused by various Colletotrichum species, such as Colletotrichum gloeosporioides and Colletotrichum cuspidosporium. In recent years, Colletotrichum aenigma was reported as a causal agent of Grape anthracnose in China and South Korea. Peroxisome is an important organelle in eukaryotes, which plays a very important role in the growth, development, and pathogenicity of several plant-pathogenic fungal species i, but it has not been reported in C. aenigma. In this work, the peroxisome of C. aenigma was labeled with a fluorescent protein, using green fluorescent protein (GFP) and red fluorescent protein (DsRED and mCherry) as reporter genes. Via Agrobacterium tumefaciens-mediated transformation (AtMT), two fluorescent fusion vectors to mark the peroxisomes, with GFP and DsRED, respectively, were introduced into a wild-type strain of C. aenigma. In the transformants, bright dots of green or red fluorescence in hyphae and spores could be seen in the strains labeled peroxisome. The nuclei labeled by the same method showed bright round fluorescent spots. In addition, we also combined fluorescent protein labeling with chemical staining to show the localization more clearly. The ideal peroxisome and nuclear fluorescence-labeled C. aenigma strain was obtained, which provided a reference for the study of its growth, development, and pathogenicity.

Identification of Colletotrichum aenigma as the new causal agent of leaf blight disease on Aucuba japonica Thunb., and screenings of effective fungicides for its sustainable management.

Aucuba japonica Thunb is an evergreen woody ornamental plant with significant economic and ecological values. It also produces aucubin, showing a variety of biological activities. It is widely planted in the southwest region of China, including karst landscape areas in Guizhou Province. In January 2022, a serious leaf blight disease was observed on the leaves of A. japonica in the outdoor gardens of Guizhou University, Guiyang, Guizhou, China. The causal agent was identified as Colletotrichum aenigma through amplification and sequencing of the internal transcribed spacer (ITS) region, translation of the chitin synthase (CHS) and actin (ACT) genes, and morphological characterizations. Koch's postulates were confirmed by its pathogenicity on healthy leaves, including re-isolation and identification. To our knowledge, this is the first report of C. aenigma causing leaf blight on A. japonica worldwide. To identify pathogen characteristics that could be utilized for future disease management, the effects of temperature and light on mycelial growth, conidia production, and conidial germination, and the effects of humidity on conidial germination were studied. Optimal temperatures for mycelial growth of C. aenigma BY827 were 25-30°C, while 15°C and 35°C were favorable for conidia production. Concurrently, alternating 10-h light and 14-h dark, proved to be beneficial for mycelial growth and conidial germination. Additionally, conidial germination was enhanced at 90% humidity. In vitro screenings of ten chemical pesticides to assess their efficacy in suppressing C. aenigma representative strain BY827. Among them, difenoconazole showed the best inhibition rate, with an EC50 (concentration for 50% of maximal effect) value of 0.0148 µg/ml. Subsequently, field experiment results showed that difenoconazole had the highest control efficiency on A. japonica leaf blight (the decreasing rate of disease incidence and decreasing rate of disease index were 44.60 and 47.75%, respectively). Interestingly, we discovered that C. aenigma BY827 may develop resistance to mancozeb, which is not reported yet among Colletotrichum spp. strains. In conclusion, our study provided new insights into the causal agent of A. japonica leaf blight, and the effective fungicides evaluated provided an important basis and potential resource for the sustainable control of A. japonica leaf blight caused by C. aenigma in the field.