Evidences of Colletotrichum fructicola Causing Anthracnose on Passiflora edulis Sims in China

Passion fruit (Passiflora edulis) is a tropical and subtropical plant that is widely cultivated in China due to its high nutritional value, unique flavor and medicinal properties. In August 2020, typical anthracnose symptoms with light brown and water-soaked lesions on Passiflora edulis Sims were observed, which result in severe economic losses. The incidence of this disease was approximately 30%. The pathogens from the infected fruit were isolated and purified by the method of tissue isolation. Morphological observations showed that the colony of isolate BXG-2 was gray to celadon and grew in concentric circles. The orange conidia appeared in the center after 14 days of incubation. The pathogenicity was verified by Koch’s postulates. The internal transcribed spacer (ITS), chitin synthase (CHS-1), actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified by relevant PCR programs. The multi-gene (ITS, GAPDH, ACT, CHS-1) phylogeny analysis confirmed that isolate BXG-2 belongs to Colletotrichum fructicola. The inhibitory effect of six synthetic fungicides on the mycelial growth of the pathogen was investigated, among which difenoconazole 10% WG showed the best inhibitory effect against C. fructicola with an EC50 value of 0.5579 mg·L−1. This is the first report of anthracnose on Passiflora edulis Sims caused by Colletotrichum fructicola in China.

First Report of Leaf Spot Caused by Colletotrichum fructicola on Myrica rubra in China

Myrica rubra is an important fruit tree with high nutritional and economic value, which is widely cultivated in multiple regions of China. In January 2021, an unknown disease which caused leaf spot with approximately 20% (n=100 investigated plants) of incidence was discovered on the leaves of M.rubra in Jiujiang City of Jiangxi Province (29.71° N, 115.97° E). The initial symptoms were small pale brown spots (1 to 2 mm diameter) on the leaves, which gradually expanded into round or irregular dark brown spots with the occurrence of the disease, and the lesion developed necrotic tissues in the center at later stages, eventually leading the leaves to chlorotic and wilted. Ten diseased leaves with typical symptoms were collected and the leaf tissue (5 × 5 mm) at junction of diseased and healthy portion were cut. The surfaces were disinfected with 75% ethanol for 45 s, 1% sodium hypochlorite for 1 min, and rinsed in sterile water for 3 times then transferred to potato dextrose agar (PDA) at 28 ± 1 ℃ for 3 days. Five fungal single isolates with similar morphology were purified from single spores. On PDA medium, the colonies initially appeared white with numerous aerial hyphae, and the center of the colony turned gray at later stages, less sporulation. While on modified czapek-dox medium (Peptone 3g, K2HPO4 1g, MgSO4·7H2O 0.5g, KCl 0.5g, FeSO4 0.01g, Maltose 30g, Agar 15g, Distilled water 1000 mL, pH=7.0), the mycelia of the colony were sparse and produced a large number of small bright orange particles (conidial masses). Conidia were single-celled, transparent, smooth-walled, 1-2 oil globule, cylindrical with slightly blunt rounded ends, 14.45-18.44 × 5.54-6.98 μm (av=16.27 μm × 6.19 μm, n=50) in size. These morphological characteristics of the pathogen were similar to the descriptions of Colletotrichum fructicola (Ruan et al, 2017; Yang et al, 2021). To further confirm the identity of the pathogen, genomic DNA from a representative isolate was extracted with DNA Extraction Kit (Yeasen, Shanghai, China), and the internal transcribed spacer (ITS), glyceraldehyde-3-phosphatedehydrogenase (GAPDH), calmodulin gene (CAL), actin (ACT) and chitin synthase 1 (CHS 1) were amplified by using the primers ITS1/ITS4 (Gardes et al, 1993), GDF/GDR (Templeton et al, 1992), CL1C/CL2C (Weir et al, 2012), ACT-512F/ACT-783R and CHS-79F/CHS-345R (Carbone et al, 1999), respectively. The PCR amplified sequences were submitted to GenBank (GenBank Accession No. ITS, MW740334; GAPDH, MW759805; CAL, MW759804; ACT, MW812384; CHS-1, MW759803) and aligned with GenBank showed 100% identity with C. fructicola (GenBank Accession No. ITS, MT355821.1 (546/546 bp); GAPDH, MT374664.1 (255/255 bp); CAL, MK681354.1 (741/741 bp); ACT, MT364655.1 (262/262 bp); CHS, MT374618.1 (271/271 bp)). Phylogenetic tree using the maximum likelihood methods with Kimura 2-parameter model and combined ITS-ACT-GAPDH-CHS-CAL concatenated sequences, bootstrap nodal support for 1000 replicates in MEGA7.0, revealed that the isolate was assigned to C. fructicola strain (ICMP 18581 and CBS 125397) (Yang et al. 2021) with 98% bootstrap support. Pathogenicities of were tested on fifteen healthy M. rubra plants (five for wounded inoculation, five for nonwounded inoculation, and five for controls) in the orchard. Twenty leaves were marked from each plant, and disinfected the surface with 75% ethanol. Ten μL spore suspension (1.0 × 106 conidia/ml) of each isolate from 7-day-old culture were inoculated on the surface of 20 needle-wounded and 20 nonwounded leaves, respectively. Healthy leaves were inoculated with sterile water as controls by the same method. All inoculated leaves were sprayed with sterile water and covered with plastic film to remained humidification. After 5 days, all the wounded leaves which were inoculated with C. fructicola showed similar symptoms to those observed on the original leaves. Symptoms of nonwounded leaves were milder than the wounded inoculated leaves, while control leaves remained healthy. Finally, the C. fructicola was re-isolated from the inoculated leaves. C. fructicola has been reported on Juglans regia, Peucedanum praeruptorum, Paris polyphylla var. Chinensis in China (Wang et al, 2017; Ma et al, 2020; Zhou et al, 2020). As far as we know, this is the first report of C. fructicola causing leaf spot on M.rubra in China. This result contributes to better understand the pathogens causing diseases of M.rubra in this region of China and develop effective control strategies.

Transcriptome Analysis of Colletotrichum fructicola Infecting Camellia oleifera Indicates That Two Distinct Geographical Fungi Groups Have Different Destructive Proliferation Capacities Related to Purine Metabolism

Anthracnose, caused by Colletotrichum spp., is a significant disease affecting oil tea (Camellia oleifera Abel.). Extensive molecular studies have demonstrated that Colletotrichum fructicola is the dominant pathogen of oil tea anthracnose in China. This study aims to investigate differences in molecular processes and regulatory genes at a late stage of infection of C. fructicola, to aid in understanding differences in pathogenic mechanisms of C. fructicola of different geographic populations. We compared the pathogenicity of C. fructicola from different populations (Wuzhishan, Hainan province, and Shaoyang, Hunan province) and gene expression of representative strains of the two populations before and after inoculation in oil tea using RNA sequencing. The results revealed that C. fructicola from Wuzhishan has a more vital ability to impact oil tea leaf tissue. Following infection with oil tea leaves, up-regulated genes in the strains from two geographic populations were associated with galactosidase activity, glutamine family amino acid metabolism, arginine, and proline metabolism. Additionally, up-regulated gene lists associated with infection by Wuzhishan strains were significantly enriched in purine metabolism pathways, while Shaoyang strains were not. These results indicate that more transcriptional and translational activity and the greater regulation of the purine metabolism pathway in the C. fructicola of the Wuzhishan strain might contribute to its stronger pathogenicity.

Leaf Blight Caused by Colletotrichum Fructicola of Large Cardamom (Amomum Subulatam Roxb.) an Important Cash Crop Grown in Sikkim, India

The full text of this preprint has been withdrawn by the authors due to author disagreement with the posting of the preprint. Therefore, the authors do not wish this work to be cited as a reference. Questions should be directed to the corresponding author.

First Report of Colletotrichum fructicola Causing Anthracnose on Indian Jujube (Ziziphus mauritiana) in Taiwan

Ziziphus mauritiana Lamarck known as Indian jujube is one of the most popular and delicious fruit crops in Taiwan. This crop is mainly planted in southern Taiwan and their fruit are harvested for providing fresh fruit. In March 2015, an anthracnose-like disease was observed on Indian jujube fruit (cv. Candied date) planted in an orchard in Yanchao District (22°46'33" N, 120°21'37" E) in Kaohsiung City. The disease was quickly distributed around the orchard after rain and caused great loss (around 40% of fruit infected). The diseased fruit would completely rot and lose its market value. Symptoms could be observed on all the developmental stages of fruit. On ripe fruit, symptoms were round, brown, water-soaked lesions covered with salmon-colored spore masses. Four fungal isolates from diseased fruit in the same orchard were collected by single spore isolation with hand-made glass needle. They were grown on potato dextrose agar (PDA) at 24 to 28°C with diffused light. All four strains produced white to gray, aerial, and cottony mycelia scattered with abundant salmon-colored conidial mass on the center of the colony on PDA. The conidia were hyaline, single celled, round cylindrical on both ends, thin walled, and the contents guttulate. The sizes of conidia were 15.2 (17.5 to 13.0) × 5.0 (5.5 to 4.5) μm (length/width ratio = 3.03, n = 40). DNA was isolated from JC1 and used for amplification of partial sequences of the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), β-tubulin 2 (TUB2), chitin synthase 1 (CHS-1), manganese-superoxide dismutase (SOD2) and the intergenic region of apn2 and MAT1-2-1 gene (ApMat) genes (Silva et al. 2012; Weir et al. 2012). A BLAST search against the NCBI database revealed that JC1 gene sequences [GenBank accession nos. MT197188 (ITS), MT199871 (GAPDH), MT199872 (ACT), MT199870 (TUB2), MT815918 (CHS-1), MT815919 (SOD2) and MT221653 (ApMat)] displayed 100.0, 100.0, 99.1, 100.0, 99.7, 99.5 and 99.8% nucleotide identity to the respective gene sequences of Colletotrichum fructicola ICMP 18581 (JX010165, JX010033, FJ907426, JX010405, JX009866, JX010327, and JQ807838). Conidial suspension (1 × 106 conidia/mL) was prepared from JC1 isolate of C. fructicola and inoculated by spraying onto detached, ripe, healthy, non-wounded and surface-disinfected jujube fruit (cv. Candied date, n = 4). Four control fruit were sprayed with sterile water. Fruit were kept in a moist chamber (greater than 90% relative humidity, 24 to 28°C) for 24 h and maintained in the lab for additional 5 days. The inoculated fruit initially showed small light-colored spots in 5 to 7 days, which eventually developed into brown, sunken, water-soaked lesions 8 to10 days after inoculation, similar to the symptoms in the orchard. C. fructicola was re-isolated from symptomatic fruit showing similar morphological characteristics to those collected from the field, thus fulfilling Koch’s postulates. No symptom was observed on fruit treated with water and no pathogen was re-isolated. The experiment was performed twice. The JC1 isolate of C. fructicola with the identification number BCRC FU31437 has been deposited at Taiwan Bioresource Collection and Research Center. This pathogen has been found in many plant species in various countries (Weir et al. 2012). To our knowledge, this is the first report of C. fructicola causing Indian jujube fruit anthracnose in Taiwan and worldwide.

First report of leaf spot caused by Colletotrichum fructicola on Dalbergia hupeana in China

Dalbergia hupeana is a kind of wood and medicinal tree widely distributed in southern China. Since 2019, a leaf spot disease was observed on the leaves of D. hupeana in Gangxia village, Luoting town in Jiangxi Province, China (28°52′53″N, 115°44′58″E). The disease incidence was estimated to be above 50%. The symptoms began as small spots that gradually expanded, developing a brown central and dark brown to black margin. The spots ranged from 4 to 6 mm in diameter. Leaf pieces (5 × 5 mm) from lesion margins were surface sterilized in 70% ethanol for 30 s followed by 2% NaOCl for 1 min and then rinsed three times with sterile water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation. Fifteen strains with similar morphological characterizations were isolated, and three representative isolates (JHT-1, JHT-2, and JHT-3) were chosen and used for further study. Colonies on PDA of three isolates were grayish-green with white edges and dark green on the reverse side. Conidia were transparent, cylindrical with rounded ends, and measured 3.6-5.3 µm × 9.5-15.2 µm (3.7 ± 0.2 × 13.6 ± 1.1 µm, n = 100). Appressoria were dark brown, globose or subcylindrical, and ranged from 6.2-9.2 µm× 5.1-6.8 µm (7.9 ± 0.4 × 5.9 ± 0.3 µm, n=100). The morphological characteristics of the three strains were consistent with the description of species in the Colletotrichum gloeosporioides complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), chitin synthase (CHS-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-tubulin 2 (TUB2) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, ACT-512F/ACT-783R, CL1/CL2, CHS-79F/CHS-345R, GDF/GDR and T1/Bt2b (Weir et al. 2012), respectively. The sequences were deposited in GenBank (Accession Nos. MZ482016 - MZ482018 for ITS; MZ463636 - MZ463638 for ACT; MZ463648- MZ463650 for CAL; MZ463639 - MZ463641 for CHS-1; MZ463642 - MZ463644 for GAPDH; MZ463645 - MZ463647 for TUB2). A neighbor-joining phylogenetic tree was constructed with MEGA 7.0 using the concatenation of multiple sequences (ITS, ACT, GAPDH, TUB2, CHS-1, CAL) (Kumar et al. 2016). According to the phylogenetic tree, three isolates fall within the Colletotrichum fructicola clade (boot support 99%). Based on morphological characteristics and phylogenetic analysis, three isolates were identified as C. fructicola. The pathogenicity of three isolates was conducted on two-yr-old seedlings (30 cm tall) of D. hupeana. Healthy leaves were wounded with a sterile needle and then inoculated with 10 μL spore suspension (106 conidia per mL). Controls were treated with sterile water. All plants were covered with transparent plastic bags and incubated in a greenhouse at 28°C with a 12 h photoperiod (relative humidity > 80%). Within five days, the inoculated leaves developed lesions similar to those observed in the field, whereas controls were asymptomatic. The experiments repeated three times showed similar results. The infection rate was 100%. C. fructicola was re-isolated from the lesions, whereas no fungus was isolated from control leaves. C. fructicola can cause leaf diseases in a variety of hosts, including Aesculus chinensis (Sun et al. 2020), Peucedanum praeruptorum (Ma et al. 2020), and Mandevilla × amabilis (Sun et al. 2020). C. brevisporum and C. gigasporum were also reported to infect Dalbergia odorifera (Chen et al. 2021; Wan et al. 2018). However, This is the first report of C. fructicola associated with leaf spot disease on D. hupeana in China. These results will help to develop effective strategies for appropriately managing this newly emerging disease.

The SNARE Protein CfVam7 Is Required for Growth, Endoplasmic Reticulum Stress Response, and Pathogenicity of Colletotrichum fructicola

The tea-oil tree Camellia oleifera is native to China and is cultivated in many parts of southern China. This plant has been grown for over 2,000 years, mainly for its high-quality cooking oil. Anthracnose is the main disease of tea-oil tree and results in a huge loss annually. Colletotrichum fructicola is a major pathogen causing anthracnose on tea-oil tree. In a previous study, we characterized that the bZIP transcription factor CfHac1 controlled the development and pathogenicity of C. fructicola. Here, we identified and characterized the function of CfVAM7 gene, which was significantly downregulated at the transcriptional level in the ΔCfhac1 strain under dithiothreitol stress. Targeted gene deletion revealed that CfVam7 is important in growth, pathogenicity, and responses to endoplasmic reticulum-related stresses. Further analysis revealed that CfVam7 is required for appressorium formation and homotypic vacuole fusion, which are important for fungal pathogen invasion. Cytological examinations revealed that CfVam7 is localized to vacuole membranes in the hyphal stage. The Phox homology (PX) and SNARE domains of CfVam7 were indispensable for normal cellular localization and biological function. Taken together, our results suggested that CfVam7-mediated vacuole membrane fusion promotes growth, stress response, and pathogenicity of C. fructicola.

Leaf Blight Caused by Colletotrichum Fructicola of Large Cardamom (Amomum Subulatam Roxb.) an Important Cash Crop Grown in Sikkim, India

Large cardamom (Amommum subulatum Roxb.) a high valued spice crop grown in Sikkim Himalaya is now facing a devastating leaf blight disease that has brought down the yield drastically. Present study was focused on identification of this major fungal pathogen based on the morphological and molecular characterization. During this study infected leaves of large cardamom with blighted appearance were collected from all the four districts of Sikkim. The pathogen was isolated using Potato Dextrose Agar (PDA) medium, incubated at 25°C. The mycelium was septate, hyaline, and 2-4 µm wide. The conidiospores were cylindrical with both ends rounded, sometimes oblong. Length and breadth were 11-12 µm and 3-4 µm, respectively. On the basis colony morphology, growth and microscopic observations, out of the total 48 samples studied Colletotrichum sp. was identified from 14 samples. Based on phylogenetic analysis of the ITS4, ITS5 and ApMAT genes and phenotypic characters (colony morphology, microscopic features) the isolate (No. LC05) isolated from the sample collected from the village Assam Linzey, East Sikkim showed 100% homology with Colletotrichum fructicola from NCBI database. The pathogenicity of C. fructicola was also confirmed during the study. The fungal culture has been deposited at the NFCCI-ARI, Pune with an accession number NFCCI 4542 and the sequences have been deposited in NCBI GenBank with accession number (ITS) MN710587, (ApMAT) MW348934 respectively. To the best of our knowledge this is the first report of C. fructicola causing blight disease of large cardamom. Also the finding is very important to improve the disease control strategies of this high valued cash crop.