Colletotrichum Gloeosporioides Species Complex Research Articles

First report of Colletotrichum henanense causing anthracnose on Chinese chestnut in the United States.

Culinary chestnut production in the United States (US) is a rapidly growing industry supporting fresh market and value-added industries. An estimated 200-400 new acres of chestnuts are planted every year in the US, with most growers east of the Rocky Mountains planting Chinese chestnut (Castanea mollissima) or Chinese chestnut hybrids. In 2018, Ohio producers of Chinese chestnut reported losses of up to 80% to blossom end rot. Symptoms were like those reported by Fowler and Berry (1958), including black spots on the chestnut shell, often at the stylar end, and blackening of the kernels. Spots covered 1-100% of the kernel, however, no signs of any pathogen were present on the shell or kernel. In 2020, cankers that were brown/black in color, sunken, and ~1 cm in length were observed on 1-yr twigs of chestnut seedlings from a nursery operation on the same farm from which the symptomatic kernels were observed. In some seedlings, distal portions of the twigs died, while in other seedlings only shoots and leaves within the cankered areas died. Black acervuli were observed erupting from the cankers. Colletotrichum spp. were isolated and cultured on potato dextrose agar from surface-sterilized tissue from kernel lesions (MLI246-21 to MLI249-21) and twig cankers (MLI250-21 and MLI251-21). All isolates produced grey aerial mycelia, pink sporodochia, and cylindrical conidia with rounded ends ranging in size from 12-20 um long by 5-8 um wide. Isolates were preliminarily identified as belonging to the Colletotrichum gloeosporioides species complex (CGSC). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, β-tubulin (TUB2) gene, and the intergenic spacer ApMat were amplified from genomic DNA and sequenced (Dowling et al. 2020). These genes are suitable for identifying species within the CGSC (Eaton et al. 2021). Sequences were submitted to the GenBank database (GAPDH OL687148 to OL687153, TUB2 OL741624 to OL741629 and ApMAT OL695914 to OL695919). BLASTn queries of NCBI GenBank showed that the GAPDH, TUB2, and ApMat sequences from all isolates had 98%, 98% and 99% identity with C. henanense isolates MT513015.1, MT513080.1, and MT512917.1 from apple (Martin et al. 2021). Representative isolates were used to demonstrate Koch's postulates and confirm pathogenicity on kernels (MLI246-21 and MLI249-21) and twigs (MLI250-21). Developing chestnuts in burs (n=4 per isolate) were gathered from the field and surface-sterilized with 70% ethanol. Nuts (n=12 per isolate) inside burs were inoculated by injecting 50uL of inoculum (~1.0 x 106 conidia/mL) directly into each kernel with a hypodermic needle and sterile syringe. Burs were incubated at room temperature in a moist chamber for 14 days. One-year-old seedlings (n=6) grown in containers were used for twig inoculations. Twig nodes (one/seedling) were surface sterilized with 70% ethanol and inoculated by wounding the stem with a sterile probe and dropping 100uL of inoculum into each wound. Seedlings were incubated in a glass house for 60 days and monitored daily for symptom development. Nuts (n=9) and twigs (n=3) were inoculated with sterile water using the same inoculation and incubation conditions to serve as negative controls. Inoculated kernels developed characteristic brown/black lesions at the wound site and inoculated twigs developed brown/black, slightly sunken cankers with acervuli. No symptoms developed on the control kernels or twigs. Fungi with the same colony, conidial, and molecular characteristics as C. henanense were re-isolated from inoculated kernels and twigs. Blossom end rot caused by Glomerella cingulata has been reported on chestnut kernels in Georgia (Fowler and Berry 1958), but this is the first report of C. henanense causing rot on kernels and twigs in the US. Since 2018, C. henanense has been isolated from infected nuts received from commercial orchards in Pennsylvania, Missouri, and Alabama. We propose to retire the name "blossom end rot" for the symptoms found in kernels and replace it with the name "chestnut anthracnose" for this disease that affects both twigs and kernels of Chinese chestnut.

First report of Colletotrichum black leaf spot on strawberry caused by Colletotrichum siamense in China

AbstractIn August 2020, a new fungal disease was observed on strawberry in Shandong Province, which showed that small spots of grayish‐black, near round without lesions and haloes on the adaxial of strawberry leaves. The morphological features of colonies and conida on PDA medium were consistent with Colletotrichum gloeosporioides species complex. The rDNA internal transcribed spacer, calmodulin, chitin synthase, and actin from the isolates were amplified and sequenced. BLAST analysis of these four genes showed 99.24‐100.00% identity with the corresponding sequences of C. siamense in GenBank. The result of phylogenetic analysis also indicated that the pathogen was identified as C. siamense. Similar symptoms were observed on the back of strawberry leaf after spraying conidial suspensions for 3 days, and C. siamense was reisolated which confirm the Koch's rule. This is the first report of new disease of black leaf spot on strawberry caused by C. siamense.

Method to detect and quantify colonization of anthracnose causal agent Colletotrichum gloeosporioides species complex in strawberry by real‐time PCR

AbstractAnthracnose, a destructive disease in strawberry worldwide, is caused by the pathogenic fungus Colletotrichum ssp.. To date, numerous studies focusing on its identification, pathogenicity and biological characteristics have been published. However, research on quantifying the colonization of this pathogen is still lacking, which is crucial to the study of anthracnose resistance. In this study, we developed a sensitive primer pair targeting the cutinase gene that is capable of distinguishing Colletotrichum gloeosporioides (C. gloeosporioides) species complex, the main species causing strawberry anthracnose in China, including C. fructicola, C. gloeosporioides, C. aenigma and C. siamense, from other Colletotrichum species and typical fungal pathogens. On this basis, we established a quantitative real‐time PCR (qRT‐PCR) assay to accurately quantify pathogen amounts from both pure cultures and infected strawberries, with detection limits of 105–101 copies. Using this method, we characterized quantitative traits of C. fructicola colonization in strawberry leaves and discovered differences in pathogen populations at different positions. Moreover, monitoring of pathogen growth during infection showed a dynamic change in the amount of C. fructicola. Finally, a comparison of the absolute and relative quantification results further proved this method's validity. Therefore, we propose that this specific PCR method enables rapid detection and quantification of C. gloeosporioides species complex colonization in strawberry and will be a useful tool for anthracnose management and strawberry resistance studies.

Identification and characterization of Colletotrichum species associated with anthracnose on persimmon in Brazil

Persimmon (Diospyros kaki) anthracnose is a major threat in production areas worldwide. Most of the studies are focused on Colletotrichum horii, but other species have been reported as well. The association of distinct Colletotrichum species present in Brazilian persimmon production regions as well as their host ranges are yet elusive. The aims of this work were to identify and characterize Colletotrichum species associated with the persimmon anthracnose. In a survey performed in four production regions of Brazil, 88.7% and 11.3% out of 231 isolates were identified as members of Colletotrichum gloeosporioides species complex (Cgc) or Colletotrichum acutatum species complex (Cac), respectively. A subset of 18 isolates were identified through multilocus phylogenetic analysis, using ITS-rDNA region and two loci, namely GAPDH and TUB2. This study revealed the presence of four species: C. horii (38.8%) and Colletotrichum fructicola (27.7%) from the Cgc and Colletotrichum nymphaeae (27.7%) and Colletotrichum melonis (5.8%), from the Cac. Additionally, 13 isolates were selected for morphological, physiological, and pathogenic analyses. Contrasting characteristics were observed among species of the Cgc and Cac complexes. The optimal temperature for mycelial growth and germination were higher for Cgc species. The percentage of appressoria melanisation also varied across complexes. All the identified species were able to cause anthracnose-like symptoms on persimmon fruit, leaves, shoots, and sepals. Colletotrichum species from persimmon were also able to infect apple and pear. The findings will support decisions to manage anthracnose of persimmon under high infection risk due to multiple host susceptibility.

Sensitivity of Colletotrichum fructicola and Colletotrichum siamense of Peach in China to Multiple Classes of Fungicides and Characterization of Pyraclostrobin-Resistant Isolates.

Anthracnose, mainly caused by Colletotrichum gloeosporioides species complex including Colletotrichum fructicola and Colletotrichum siamense, is a devastating disease of peach. Chemical control has been widely used for years, but management failures have increased with the commonly used fungicides. Therefore, screening of sensitivity of Colletotrichum spp. to fungicides with different modes of action is needed to make proper management strategies for peach anthracnose. In this study, the sensitivity of 80 isolates of C. fructicola and C. siamense was screened for pyraclostrobin, procymidone, prochloraz, and fludioxonil based on mycelial growth inhibition at discriminatory doses. Results showed that C. fructicola and C. siamense isolates were highly resistant to procymidone and fludioxonil with 100% resistance frequencies to both fungicides, but sensitive to prochloraz, i.e., no resistant isolates were found. For pyraclostrobin, 74% of C. fructicola isolates showed high resistance, 26% showed low resistance, and all of the C. siamense isolates showed low resistance. No positive cross-resistance was observed between pyraclostrobin and azoxystrobin even when they are members of the same quinone outside inhibitor (QoI) fungicide group or between pyraclostrobin and non-QoIs. Resistant isolates to QoI fungicides were evaluated for the fitness penalty. Results showed that no significant differences except for the mycelial growth rates that were detected between high- and low-resistance isolates of C. fructicola. Molecular characterization of the Cyt b gene revealed that the G143A point mutation was the determinant of the high resistance in C. fructicola. This study demonstrated the resistance status of C. fructicola and C. siamense to different fungicides and briefly discussed implications of that resistance. Demethylation inhibitor fungicides were found to be the best option among the different chemicals studied here, to control peach anthracnose in China.

First report that Colletotrichum aenigma causes leaf spots on Aquilaria sinensis in China.

Aquilaria sinensis (Lour.) Spreng, also known as eaglewood, belongs to the Thymelaeaceae family and has a considerably high medicinal value. It has been enlisted as the class II national key protective plant. In June 2019, about 15 percent of A. sinensis treelets in a forest area of China's Hainan province were observed to have the anthracnose symptoms. The diseased spots on leaves of A. sinensis treelets were usually round or irregular with pale yellow edges. The color of the center of the lesion was firstly light brown and then black or yellowish-brown. Small pieces of tissue from the edge of the leaf spots were surface sterilized in 75% alcohol for the 60s, washed twice with sterile distilled water, and then cultivated at 28 °C in darkness on potato dextrose agar (PDA) medium. One fungus was systematically isolated to get pure cultures. The culturing of the three isolates was carried out in PDA media at 28 °C for a week. The average diameter of the collateral colony was 6.80 ±0.60 cm. Initially, the fungal colonies were white aerial mycelium and the central area of the colonies slowly turned jacinth. After seven days, the central mycelium turns grayish-green and the colonies' undersurfaces were grey to white. The colony's surfaces were fluffy and round with smooth edges. Conidia were cylindrical, smooth, and transparent, with a slight indentation in the middle and uneven distribution of small particles inside, 12.5-20.6×3.5-6.8 µm (ave=15.9±1.40×5.18±1.07, n=50). Appressoria were typically elliptic or irregular and brown to dark brown. The isolates were characterized as Colletotrichum gloeosporioides species complex on the basis of the conidial morphology and culture representation, (Deng et al. 2017; Weir et al. 2012). To further verify the identification of the species, CX-0301, the isolated representative strains were extracted for genomic DNA. mating type 1-2-1 (Mat-1-2-1) ApMat, actin (ACT) gene, chitin synthase (CHS), and beta-tubulin (TUB2) gene were amplified using the primer pairs VcaMat-5F/VcaMat-5R, ACT-512F/ACT-783R, CHS-1-79F/CHS-1-354R, and TUB2-T1/Bt2b, respectively (Damm et al. 2012; Du et al. 2005). The homologous sequences of MN310694, MN310693, MN310692, and MN310691 were submitted to GenBank. These genes have ≥a 97% sequence similarity to the genes of Colletotrichum aenigma (MG717319.1, MG717317.1, MH476565.1, MH853679.1, respectively) in GenBank. These morphological and molecular characteristics identified that the pathogen is C. aenigma. (Weir et al. 2012). To further verify the isolated pathogen, the pathogenicity test was performed on uninfected healthy 2-year-old eaglewood seedlings. The conidial suspension (1×106 conidia/ml) of 5ml was sprayed on both surfaces of 10 leaves of plants of the same age and height and the controls were treated solely with distilled water (Deng et al. 2017). Upon completion of inoculation, plants were kept under greenhouse conditions with an assigned temperature of 28 ± 2°C while keeping relative humidity to 90% on a 12-h fluorescent light/dark regime. Anthracnose-like symptoms were observed 6 days postinoculation. The control plant tissues remained healthy. Follow up reisolation of C. enigma culture was obtained in PDA agar plates from leaf infected lesions, and the morphological features were found to be consistent with that of CX-0301 isolate, satisfying Koch's postulates. Based on the characterized information, it is the first report of Colletotrichum aenigma responsible for causing leaf spots on Aquilaria sinensis in China. Thereby, this provides a theoretical reference for the research and control of anthracnose on A. sinensis.

Diseases and Insect Pests associated with Cashew (Anacardium occidentale L.) Orchards in Ghana

Cashew (Anacardium occidentale L.), a recently recognized cash crop in Ghana, is an economically export oriented crop that plays a helping role in the Ghanaian economy in diverse ways. Owing to its importance, surveys were conducted in some major cashew producing communities in Dormaa and Berekum West District of the Bono region to assess the problems of insect pests and diseases associated with this economic tree across the study area. Diseased samples showing varying degrees of symptoms were taken for isolation and identification using morphological method. Insect pests were also assessed and identified. The results showed Colletotrichum gloeosporioides species complex to be associated with anthracnose, leaf lesions or spots, gummosis of twigs and stems and regressive die-back; Lasiodiplodia theobromae was found to cause stem and twig gummosis, blight, die-back of twigs and inflorescence; Pestalotia sp. was associated with Pestalotia leaf spot. Others, such as Penicillium sp., powdery mildew, Curvularia lunata, Cephaleuros sp. (red rust- algal leaf spot), mushroom and lichens were also found to be associated with the cashew orchards in the study area. Anthracnose, gummosis, algal leaf, and stem spots (red rust) and cashew kernel infection by Curvularia lunata constituted the major diseases of cashew in ascending order in the study area. The insect pests identified included Oecophylla smaragdina, Anoplocne miscurvipeson, Pseudotheraptus devastans, Pachnoda cordata, Pachnoda marginata, Helopeltis bug, Helopeltis schoutedeni, Planococcus sp., Lamida moncusalis, Odontotermes sp., Aphis sp., Analeptes trifasciata. Some unidentified pathogens, pests and other abnormalities were also observed.

Identification of the Colletotrichum species associated with mango diseases and a universal LAMP detection method for C. gloeosporioides species complex

Abstract: The Colletotrichum gloeosporioides species complex contains plant pathogens linked to Anthracnose diseases afflicting various crops. In this study, we designed a loop-mediated isothermal amplification assay (LAMP) primer set based on calmodulin gene coding region sequences from taxonomically authorized isolates of species from this complex to rapidly detect the presence of fungi associated with Anthracnose diseases. This test can be employed at any point between cultivation and sale. Moreover, we examined the specificity and detectable range of this primer set using isolates selected from species of the genus Colletotrichum. This test was able to specifically detect members of the C. gloeosporioides species complex, including C. gloeosporioides, C. aotearoa, C. fructicola, C. horii, C. kahawae, C. musae, C. siamense, C. theobromicola, and C. tropicale. Key Words: Anthracnose, diagnosis, phylogeny, plant disease

First Report of Colletotrichum gloeosporioides Causing Anthracnose on Pepper in Shaanxi Province, China.

Pepper (Capsicum annuum L.) is an important solanaceous vegetable crop, with high nutritional and economic value. However, it is susceptible to Colletotrichum spp. infection during its growth and development, which seriously affects production yield and quality. Chili anthracnose, caused by Colletotrichum spp., is one of the most destructive diseases of pepper. In August 2020, chili anthracnose was observed with widespread distribution in the horticulture field of Northwest A&F University (34.16° N, 108.04° E) in Shaanxi Province, China. Approximately 60% of the pepper plants had disease symptoms typical of anthracnose. Lesions on pepper fruits were dark, circular, sunken, and necrotic, with the presence of orange to pink conidial masses (Figure S1A). To perform fungal isolation, the tissue at the lesion margin was cut from eight symptomatic fruits, surface disinfested with 75% ethanol for 30 s, and 2% NaClO for 1 min, then rinsed three times with sterile distilled water and dried on sterile filter paper. The tissues were placed on potato dextrose agar (PDA) and incubated at 28 ºC in the dark. After 3 days, hyphae growing from tissue of each lesion were recultured on PDA (Liu et al. 2016). A representative single-spore isolate (NWAFU2) was used for morphological characterization, molecular analysis, phylogenetic analysis, and pathogenicity tests. NWAFU2 colonies had gray-white aerial mycelium, and the reverse side of the colonies was dark gray to light yellow after 10-days growth on PDA (Figure S1B-C). Conidia were cylindrical, aseptate, with obtuse to slightly rounded ends, and measured 10.1 to 16.9 (length) × 4.7 to 7.0 (width) μm (n=50) (Figure S1D). Based on morphological features, the isolate was consistent with the description of C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted using a CTAB method and the internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and partial sequences of actin (ACT) genes were amplified and sequenced using primers ITS1F/ITS4, GDF1/GDR1 and ACT-512F/ACT-783R, respectively (Dowling et al. 2020). Using the BLAST, ITS, ACT, GAPDH gene sequences (GenBank accession nos. MW258690, MW258691 and MW258692, respectively) were 100%, 100% and 98.19% identical to ZJL-4 of C. gloeosporioides (GenBank accession nos. MN075757, MN058142 and MN075666, respectively). Phylogenetic analysis was conducted using MEGA-X (Version 10.0) based on the concatenated sequences of published ITS, ACT and GAPDH for Colletotrichum species using Neighbor-Joining algorithm. The identified isolate (NWAFU2) was closely related to C. gloeosporioides (Figure S2). To confirm the pathogenicity, ten healthy pepper fruits were surface-sterilized and 2 μL of conidial suspension (1×106 conidia/mL) was injected the surface of pepper. Five fruits were inoculated with 2μL sterile distilled water as controls. After inoculation, the fruits were kept in a moist chamber at 28°C in the dark. The experiment was repeated three times. Anthracnose symptoms similar to those observed in the field, were observed 7 days after inoculation (Figure S1F) and control fruits remained healthy. A similarly inoculated detached leaf assay resulted in water-soaked lesions 3 days after inoculation. C. gloeosporioides was reisolated from the infected pepper fruits, fulfilling Koch's postulates. C. gloeosporioides has been reported to cause chili anthracnose in Sichuan Province, China (de Silva et al. 2019; Liu et al. 2016). However, Shaanxi is one of the main pepper producing areas in china and it is geographically distinct from Sichuan; its climate and environmental conditions are different from Sichuan. Knowledge that C. gloeosporioides causes chili anthracnose of pepper in Shaanxi province, China may aid in the selection of appropriate management tactics for this disease. Reference: de Silva, D. D., Groenewald, J. Z., Crous, P. W., Ades, P. K., Nasruddin, A., Mongkolporn, O., and Taylor, P. W. J. 2019. Identification, prevalence and pathogenicity of Colletotrichum species causing anthracnose of Capsicum annuum in Asia. IMA Fungus 10:8. Dowling, M., Peres, N., Villani, S., and Schnabel, G. 2020. Managing Colletotrichum on Fruit Crops: A "Complex" Challenge. Plant Dis 104:2301-2316. Liu, F. L., Tang, G. T., Zheng, X. J., Li, Y., Sun, X. F., Qi, X. B., Zhou, Y., Xu, J., Chen, H. B., Chang, X. L., Zhang, S. R., and Gong, G. S. 2016. Molecular and phenotypic characterization of Colletotrichum species associated with anthracnose disease in peppers from Sichuan Province, China. Sci Rep 6. Weir, B. S., Johnston, P. R., and Damm, U. 2012. The Colletotrichum gloeosporioides species complex. Stud Mycol 73:115-180.

First report of leaf spot caused by Colletotrichum siamense on Michelia alba in China.

Michelia alba (common name: whitechampaca), native to Indonesia, is a preciously ornamental and medicinal plant in the west and southeast of China and widely distributed in Nanning, Guangxi, China (Hou et al. 2018). In May 2020, a foliar disease of M. alba was observed in Nanning (22°51' N; 108°17' E), Guangxi, China, present on ca. 20-30% of the leaves. The disease began to develop from the margins of leaves in most cases. The symptoms recorded were light yellow spots, which gradually developed into ellipsoidal to irregular brown spots, surrounded by a wide yellow halo. The spots gradually enlarged in size and became grey-brown, with the dimension of 3.5 × 2.8 to 11.0 × 3.5 cm, even more than half of leaf area. In the later stage of infection, these spots coalesced resulting in necrosis and early shedding of the leaves. Sometimes black acervuli were observed on some lesions. For isolation of the fungus, ten symptomatic leaves were randomly sampled from five trees and washed with sterile water. Small pieces of infected tissue (about 4 mm2) were surface disinfected in 75% alcohol for 30 s and in 0.1% aqueous solution of mercury chloride for 1 min. Finally these tissue pieces were rinsed three times with sterile water, plated on potato dextrose agar (PDA) and then incubated for 7 days at 28℃ with a photoperiod of 12 h. Fifteen strains with similar morphological characterizations were isolated, and five representative isolates (BL-1 to BL-5) were purified. These cultures gave rise to grey-white colonies with bright orange conidial masses with contained one-celled, hyaline, guttulate conidia, measuring 12.68-20.70 × 4.27-7.84 µm (average 15.36 × 5.35 µm, n=100). Appressoria formed from conidia were brown, ellipsoidal or inverted trapezoid and measured 6.36-12.13 × 5.07-7.39 µm (average 8.29 × 6.36 µm, n=30). These morphological characteristics were similar to those of the Colletotrichum gloeosporioides species complex (Weir et al. 2012). To confirm identification, genomic DNA from mycelium of these five isolates was extracted, and the sequence of internal transcribed spacer (ITS), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), calmodulin (CAL) and β-tubulin (TUB2) were amplified (Zhang et al. 2020), and the GenBank accession numbers for the sequences were MW186173 to MW186177 (ITS), MW161290 to 161294 (CHS-1), MW161295 to MW161299 (GAPDH), MW161285 to 161289 (ACT), MW084710 to 084714 (CAL) and MW161300 to MW161304 (TUB2). The phylogenetic tree of six combined genes of the five isolates clustered with Colletotrichum siamense strains (CBS 125378, ICMP 17795 and ICMP 18121). Therefore, the isolates were identified as C. siamense. Five isolates (BL-1 to BL-5) were tested for pathogenicity. Wounded and unwounded detached healthy leaves were inoculated using mycelial discs (5 mm in diameter) and conidial suspensions (with the concentration of 1 × 105 conidia/ml) at the same time, incubated in a growth chamber at 25-30℃ (85-90% relative humidity, with a photoperiod of 12 h). Three leaves (wounded left half blade and unwounded right half blade) were inoculated with different methods for each isolate, and the tests were repeated three times. Four days after inoculation, leaf spots were observed on all wounded leaves, while 5-10% of the unwounded leaves showed lesions. Control leaves inoculated with PDA discs and sterile water remained symptomless. Colletotrichum. siamense was re-isolated from the lesions, confirming Koch's postulates. At least 60 plant species have been reported to be infected by C. siamense worldwide (Ji et al. 2019). To our knowledge, this is the first report of C. siamense causing leaf spot on M. albain China.

First Report of Anthracnose Caused by Colletotrichum siamense and C. fructicola of Camellia chrysantha in China.

Camellia chrysantha (Hu) Tuyama, belonging to the Theaceae family, is famous for its large size and golden yellow flowers, which has high ornamental and health care functions (Mo et al. 2013). Anthracnose is one of the most important fungal diseases worldwide, causing serious economic losses to many plants. In October 2019, severe anthracnose symptoms were observed on the leaves of C. chrysantha in a 0.6 hectare field with 15-20% disease incidence in Fangchenggang city, Guangxi Zhuang Autonomous Region of China. Diseased leaves initially appeared irregular chlorotic spots, which afterwards enlarged and coalesced. Finally, the spots became dark brown or black, sunken lesions (8-22 mm in diameter), and covered with plenty of acervuli. For pathogen isolation, the leaf lesions were cut into small tissue pieces (5 mm×5 mm), disinfected by 0.3% sodium hypochlorite for 2 min and 70% ethanol for 40 s, rinsed in sterile distilled water, and then incubated at 28°C on potato dextrose agar (PDA) plates. A total of 7 fungal isolates with whitish to light grey, dense colonies were recovered at 5 days. These isolates were tentatively identified as belonging to Colletotrichum gloeosporioides species complex through morphological and cultural characters (Weir et al. 2012). The conidia were nonseptate, cylindrical with obtuse to rounded ends, 13.9 to 18.3 (average 16.1) μm × 4.5 to 6.2 (average 5.4) μm (n = 50). For further precise identification, the 7 Colletotrichum isolates were analyzed using partial sequences of genomic loci including the internal transcribed spacer (ITS), β-tubulin (TUB), calmodulin (CAL), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), and the mating type locus MAT1-2 (ApMat) genes (Liu et al. 2015). The amplification sequences were compared with the sequences registered in the GenBank database based on nucleotide similarity. The above sequences of 4 isolates (JZB-PF4232, JZB-PF2231, JZB-PF42 and JZB-PF22) had 99-100% identity to the sequences of Colletotrichum siamense strains retrieved from GenBank, while the sequences of the other 3 isolates (JZB-PF3231, JZB-PF32 and JZB-PF41) showed over 99% identity with those of the C. fructicola strains. All the sequences were deposited in GenBank with accession number MT708987 to MT709007, MW149430 to MW149433, and MW142259 to MW142282. A multi-loci phylogenetic analysis of the concatenated sequences of ITS, TUB, CAL, ACT, GAPDH, GS and ApMat genes placed the 4 isolates described above in the C. siamense clade, while the other 3 isolates was attribute to the C. fructicola clade. Pathogenicity tests were conducted on 7 healthy 2-year-old C. chrysantha seedlings (cv. Fangpu), consisted of 21 wounded leaves made by a sterile needle, with 3 leaves per seedling. Artificial inoculations were performed by treating each seedling with 20 µl of spore suspension (106 conidia/ml) of each isolate. Leaves of seedlings treated with sterilized water under the same conditions served as controls. The experiment was repeated three times. All the seedlings were covered with plastic bags to maintain high humidity (90% RH) and placed in a greenhouse kept at 25°C with a 16 h light / 8 h dark photoperiod. After 8 days, the inoculated leaves of C. chrysantha plants developed typical dark brown or black lesions, similar to the symptoms in the field, whereas controls remained symptomless. Koch's postulates were fulfilled by re-isolation of the same fungi from symptomatic inoculated leaves, identification confirmed by morphological and molecular characteristics, respectively. C. siamense and C. fructicola have been found to cause anthracnose on Camellia sinensis (Wang et al. 2016; Shi et al. 2018). C. fructicola has also been reported to cause anthracnose on Citrus sinensis in China (Hu et al. 2019). To our knowledge, this is the first report of C. siamense and C. fructicola causing anthracnose on C. chrysantha in China.

Telomeres and a repeat-rich chromosome encode effector gene clusters in plant pathogenic Colletotrichum fungi.

Members of the Colletotrichum gloeosporioides species complex are causal agents of anthracnose in many commercially important plants. Closely related strains have different levels of pathogenicity on hosts despite their close phylogenetic relationship. To gain insight into the genetics underlying these differences, we generated and annotated whole-genome assemblies of multiple isolates of C. fructicola (Cf) and C. siamense (Cs), as well as three previously unsequenced species, C. aenigma (Ca), C. tropicale and C. viniferum with different pathogenicity on strawberry. Based on comparative genomics, we identified accessory regions with a high degree of conservation in strawberry-pathogenic Cf, Cs and Ca strains. These regions encode homologs of pathogenicity-related genes known as effectors, organized in syntenic gene clusters, with copy number variations in different strains of Cf, Cs and Ca. Analysis of highly contiguous assemblies of Cf, Cs and Ca revealed the association of related accessory effector gene clusters with telomeres and repeat-rich chromosomes and provided evidence of exchange between these two genomic compartments. In addition, expression analysis indicated that orthologues in syntenic gene clusters showed a tendency for correlated gene expression during infection. These data provide insight into mechanisms by which Colletotrichum genomes evolve, acquire and organize effectors.

First Report of Colletotrichum siamense Causing Anthracnose on Zinnia elegans Jacq. in China.

Zinnia elegans (syn. Zinnia violacea), known as common zinnia, is one of the most spectacular ornamental plants in the family Asteraceae. Zinnia plants are widely cultivated in China for their impressive range in flower colours and profuse bloom over a long period. In April 2019, Zinnia plants grown in Ningbo Botanical Garden (29°56'57″N, 121°36'20″E) were found to have many circular necrotic lesions. In the early infection stage, the lesions appeared as small circular specks which developed later into large spots (15 to 32 mm diameter). Typical symptoms appeared to be grayish white centers with a chlorotic edges and disease incidence reached approximately 80% of plants in the affected field. Moreover, the growth of Zinnia plants was seriously affected by the disease. To identify the causative pathogen associated with the disease, 10 symptomatic leaves were collected from ten different Zinnia plants. Leaf tissues were cut from the lesion margins, surface sterilized with 75% ethanol for 30 seconds and rinsed three times in sterile distilled water. The leaf tissues were then dipped into 10% sodium hypochlorite for 2-3 minutes, washed three times in distilled water and dried on a sterile filter paper. After drying, the surface-sterilized leaf discs were transferred to potato dextrose agar (PDA) plates and incubated at 28°C for 2 to 3 days under the 12 h photoperiod. A total of ten pure fungal isolates were obtained and all the isolates displayed the same colony structure. Afterwards, three pure strains were randomly selected (F1, F3 and F5) for further study. The fungal colonies showed gray to brownish aerial mycelia with pink-colored masses of conidia. Conidia were one-celled, hyaline, cylindrical to subcylindrical, spindle-shaped with obtuse ends, measuring from 15.6 to 17.3 × 4.6 to 5.1 μm with both ends rounded. These morphological characteristics were consistent with the description of Colletotrichum gloeosporioides complex (Weir et al. 2012). The identity of a representative isolate, F3, was confirmed by a multilocus approach. Genomic DAN of isolate F3 was extracted and partial sequences of actin (ACT), chitin synthase (CHS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal internal transcribed spacer (ITS), manganese-superoxide dismutase (SOD2) , glutamine synthatase (GS), beta-tubulin (TUB2) and calmodulin (CAL) were amplified and sequenced as previously described (Weir et al. 2012). These nucleotide sequences were deposited in GenBank (accession MN972436 to MN972440, and MT266559 to MT266561; all sequences in FASTA format are shown (Supplementary S1). BLAST analysis of ITS, ACT, CHS, GAPDH and GS sequences from the F3 isolate revealed similarity to C. gloeosporioides voucher strain ZH01 with 100%, 100%，99%, 99% and 99% identity, respectively. SOD, TUB2 and CAL sequences showed similarity to C. siamense with 100%, 100% and 100% identity, respectively. The phylogenetic trees were constructed by Maximum Likelihood method (ML) using JTT model implemented in the MEGA 7. Results inferred from the concatenated sequences (ACT, CHS, GAPDH, ITS, SOD, GS, TUB2 and CAL) placed the isolate F3 within the C. siamense cluster (Supplementary S2). To confirm pathogenicity of the fungus, Koch's postulates were conducted by spraying 20 Zinnia plants (60-day-old) with a 1 × 106 conidia/ml suspension. Plants were maintained in the growth chamber at 25°C and 85% relative humidity. After 10 to 15 days, symptoms were observed on all inoculated leaves and resembled those observed in the field, whereas the control plants remained asymptomatic. Here, C. siamense was isolated only from the infected Zinnia leaves and identified by morphological and gene sequencing analyses. C. siamense has been reported in many crops in China (Yang et al. 2019; Chen et al. 2019; Wang et al. 2019). However, to our knowledge, this is the first report of anthracnose caused by C. siamense on Zinnia elegans in China. References Chen, X., Wang, T., Guo, H., Zhu, P. K., and Xu, L. 2019. First report of anthracnose of Camellia sasanqua caused by Colletotrichum siamense in China. Plant Dis. 103:1423-1423. Wang, Y., Qin, H. Y., Liu, Y. X., Fan, S. T., Sun, D., Yang, Y. M., Li, C. Y., and Ai, J. 2019. First report of anthracnose caused by Colletotrichum siamense on Actinidia arguta in China. Plant Dis. 103:372-373. Weir, B. S., Johnston, P. R., and Damm, U. 2012. The Colletotrichum gloeosporioides species complex. Stud. Mycol. 73: 115-180. Yang, S., Wang, H. X., Yi, Y. J., and Tan, L. L. 2019. First report that Colletotrichum siamense causes leaf spots on Camellia japonica in China. Plant Dis. 103:2127-2127.

Etiology of Cyclocarya paliurus Anthracnose in Jiangsu Province, China.

Cyclocarya paliurus is an extremely valuable and multifunctional tree species whose leaves have traditionally been used in used in medicine or as a medicinal tea in China. In recent years, anthracnose has been frequently observed on young leaves of C. paliurus in several nurseries located in Jiangsu Province, resulting in great yield and quality losses. To date, no information is available about the prevalence of C. paliurus anthracnose in China. The main purpose of the present study was to characterize the etiology of C. paliurus anthracnose. Phylogenetic analysis of the eight-loci concatenated dataset revealed that all 44 single-spore Colletotrichum isolates belonged to three species in the Colletotrichum gloeosporioides species complex, namely, Colletotrichum aenigma, Colletotrichum fructicola, and C. gloeosporioides sensu stricto. Phenotypic features, including the colony appearance and the morphology of conidia, appressoria, and ascospores, were consistent with the phylogenetic grouping. Virulence tests validated that the three Colletotrichum species could cause typical symptoms of anthracnose on C. paliurus leaves, similar to those observed in the field. The optimum mycelial growth temperature ranged from 25 to 30°C for all representative isolates, while C. gloeosporioides s. s. isolates exhibited greater tolerance to high temperature (40°C). Fungicide sensitivity assays indicated that all three Colletotrichum species were sensitive to tetramycin, which may be a potential alternative for the management of C. paliurus anthracnose. To our knowledge, this study provides the first report of C. aenigma, C. fructicola, and C. gloeosporioides s. s. causing C. paliurus anthracnose in China as well as in the world.

First Report of Colletotrichum aenigma Causing Apple Glomerella Leaf Spot on the Granny Smith Cultivar in China.

Glomerella leaf spot (GLS) is a devastating fungal disease causing pre-mature defoliation on apple (Malus domestica). It was first reported in 1970s and since then has been reported in North America, South America and Asia. GLS disease is caused by Colletotrichum fungi and the pathogens are genetically diverse, encompassing at least nine species belonging to three species complexes (Velho et al. 2018). In August 2018, disease with sudden leaf necrosis symptom, typical of GLS symptom appearance, occurred in a Granny Smith orchard in Wugong county, China, over 70% tree leaves bared brown and necrotic lesions. Small leaf tissues (3-4 mm2) cut from lesion margins were surface sterilized for 30 s in 3% NaClO and 30 s in 75% ethanol, followed by rinsing three times in sterile water before transferring onto potato dextrose agar (PDA) plates (25 ± 2°C). Seven isolates were obtained, all producing round cottony colonies on PDA, being white to pale on the upper side and dark green on the reverse side. Conidia were single-celled, cylindrical and transparent (17.33 ± 1.29 × 5.11 ± 0.77 μm, n=50). Appressoria were single-celled, thick-walled, dark brown, oval or irregular shaped, sometimes lobed (9.07 ± 0.88 × 6.66 ± 0.33 μm, n=50). The morphological and cultural characteristics of the fungal isolates matched the descriptions of Colletotrichum aenigma (Weir et al. 2012). To confirm the species identity, genomic DNAs were isolated from two representative isolates (QSG1 and QSY1), and the internal transcribed spacer (ITS), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUB2), manganese-superoxide dismutase (SOD2), chitin synthase (CHS), and calmodulin (CAL) regions were amplified by PCR using reported primers (Weir et al. 2012). The sequences were deposited in GenBank (Accession Nos. MT872061, MT873580, MT873581, MT888183, MT888185, MT888187 and MT888189 for QSG1, and MT834933, MT835166, MT873579, MT888182, MT888184, MT888186 and MT888188 for QSY1). BLASTn search against GenBank nr database showed that ITS sequences of the two strains showed high nucleotide identity (over 99%) to sequences derived from the Colletotrichum gloeosporioides species complex (CGSC). Further concatenated phylogenetic analysis with reported CGSC strains (Weir et al. 2012) placed QSG1 and QSY1 in the clade of C. aenigma. To fulfill Koch's postulates, field pathogenicity test was performed. The experiment was performed in an orchard located in Yangling in September 2020, with the daily average temperature ranging between 15 - 20 ℃. Healthy 'Granny Smith' leaves were surface sterilized with 70% alcohol and inoculated with conidial suspension (106 conidia/mL) using cotton swabs. For each isolate, 10 leaf inoculations were performed. Inoculation with distilled water served as a negative control. Inoculated leaves were covered with plastic bags to maintain high humidity and the bags were removed at 48 hours post inoculation (hpi). Conidium-inoculated leaves started to exhibit GLS-resembling necrotic lesions from 5 dpi onward. The lesion extent, however, varied among inoculated leaves, ranging from blurry, small-sized lesions to blight of entire leaf. In contrast with conidium inoculations, water-inoculated leaves remained asymptomatic until 14 dpi. Re-isolated fungi from the symptomic leaf tissues were identical to C. aenigma in morphological appearance. Taken together, this is the first report of GLS on 'Granny Smith' apple, and the first report of C. aenigma causing apple GLS in China. This information should provide important guideline for developing field control practices of GLS.

Colletotrichum Gloeosporioides Species Complex: Pathogen Causing Anthracnose, Gummosis and Die-Back Diseases of Cashew (Anacardium Occidentale L.) In Ghana

A survey was conducted in twenty-five cashew (Anacardium occidentale) orchards in five communities in the Dormaa-Central Municipality of Bono Region of Ghana to assess the incidence and severity of anthracnose, gummosis and die-back diseases on cashew. Cashew diseased samples of leaves, stem, inflorescences, twigs, flowers, nuts and apples showing symptoms (e. g. small, water-soaked, circular or irregular yellow, dark or brown spots or lesions on leaves, fruits and flowers, sunken surface, especially on the apples, blight, gum exudates) were collected for isolation of presumptive causative organism. The pathogen was isolated after disinfecting the excised diseased pieces in 70% ethanol, plated on potato dextrose agar (PDA) and incubated at 28 oC for 3 to 7 days. The identity of the putative pathogen was morphologically and culturally confirmed as belonging to Colletotrichum gloeosporioides species complex using standard mycological identification protocols. The pathogen had varied conidia sizes of between 9-15 up to 20 μm in length and diameter of 3-6 μm. The conidia were straight and cylindrically shaped with rounded or obtuse ends. The septate mycelium was whitish-grey, velvety and cotton-like in appearance from the top. The results confirmed the presence of the pathogen in the orchards with incidence ranging from 6.9% and 14.0% for gummosis and averaged 22.9% for anthracnose infected orchards. The result of the pathogenicity test confirmed the isolates to be pathogenic on inoculated cashew seedlings and were consistently re-isolated, thereby establishing the pathogen as the true causal agent of the said diseases in cashew trees and thus completed the Koch’s postulate.

Baseline sensitivity of penthiopyrad against Colletotrichum gloeosporioides species complex and its efficacy for the control of Colletotrichum leaf disease in rubber tree

Colletotrichum leaf disease (CLD), mainly caused by the Colletotrichum gloeosporioides species complex (CGSC), is a serious disease of rubber tree in most rubber-growing countries. Few fungicides are available for rubber farmers to manage this disease. To control CLD effectively, it is important to test the baseline sensitivity and efficacy of fungicides against CGSC isolates. Penthiopyrad is a new succinate dehydrogenase inhibitor (SDHI) that has been registered for use in many countries. In this study, baseline sensitivity to penthiopyrad was established by mycelial growth methods using 91 CGSC isolates collected from rubber trees in China. The EC50 values of penthiopyrad ranged from 0.243 to 3.377 mg/L with a mean EC50 value of 1.685 mg/L. Penthiopyrad greatly decreased the mycelial biomass and increased cell membrane permeability of CGSC isolates. The mycelia were severely distorted, and the number of top branches increased with penthiopyrad treatment. Penthiopyrad inhibited succinate dehydrogenase activity, causing a decrease in the ATP content of CGSC isolates, and significantly inhibited conidia germination and appressorium formation on rubber tree leaves. The protective and curative efficacy of 100 mg/L penthiopyrad reached up to 98.53% and 66.79% against CLD, respectively. Our results demonstrated the high inhibitory activity of penthiopyrad against CGSC isolates and its potential for CLD management on rubber tree.

First Report of Colletotrichum siamense and C. gloeosporioides Causing Anthracnose of Citrus spp. in Mexico.

Citrus anthracnose, caused by Colletotrichum spp., is a major disease in many citrus-growing regions of the world. During the spring of 2019, symptoms of petal necrosis and necrotic lesions on fruits were detected on Mexican lime (Citrus aurantifolia), sweet orange (Citrus sinensis), and grapefruit (Citrus paradisi) trees in three commercial orchards distributed in northern Sinaloa (El Fuerte and Ahome municipalities), Mexico. Colletotrichum-like colonies were consistently isolated on potato dextrose agar (PDA) medium from symptomatic petals and fruits, and 30 monoconidial isolates (10 per orchard) were obtained. Five isolates were selected as representative for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The isolates were designated as FAVF355-FAVF359 and were deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agronomy of El Fuerte Valley at the Autonomous University of Sinaloa (Mexico). Colonies grown on PDA at 25ºC were cottony, dense, with grayish white aerial mycelium and with pink conidial masses. Conidia (n= 100) were cylindrical, hyaline, aseptate, 13.7 to 18.8 × 4.3 to 5.8 μm, with both ends rounded. Based on morphological features, the five isolates were tentatively identified in the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For molecular identification, total DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin (TUB2) genes were amplified by PCR (Weir et al. 2012), and sequenced. A phylogenetic tree based on Bayesian inference for species belonging to the C. gloeosporioides species complex was constructed. The multilocus phylogenetic analysis distinguished the isolates FAVF355-FAVF357 as C. gloeosporioides sensu stricto and the isolates FAVF358-FAVF359 as C. siamense. The sequences were deposited in GenBank (accession numbers ITS: MT850050-MT850054; ACT: MT834528-MT834532; GAPDH: MT855979-MT855982; TUB2: MT834533-MT834536). Pathogenicity of the five isolates was verified on healthy fruits of their original host species. Five fruits per isolate were inoculated using the colonized agar plug method. Fruits were wounded with a sterile toothpick and mycelial plugs (5 mm in diameter) removed from the margin of a 6-days-old culture were placed onto three wound sites in each fruit. Non-colonized agar plugs were placed on the wounds of 10 fruits used as the control. The fruits were kept in a moist chamber at 25°C for 8 days. The experiment was repeated twice. All inoculated fruits developed circular and necrotic lesions 6 days after inoculation, whereas the control fruits remained symptomless. The fungi were consistently re-isolated from the diseased fruits and were morphologically identical to that originally inoculated, fulfilling Koch´s postulates. To date, only C. gloeosporioides sensu lato and C. acutatum sensu lato has been associated with sweet orange and Mexican lime in Mexico (Farr and Rossman 2020), whereas C. gloeosporioides sensu stricto has been recently recorded in a different area (Iguala, Guerrero) of Mexico (Cruz-Lagunas et al. 2020). To our knowledge, this is the first report of C. gloeosporioides sensu stricto causing anthracnose on sweet orange, and of C. siamense on Mexican lime in Mexico, as well as C. gloeosporioides s. s. causing disease on grapefruit in Sinaloa, Mexico.

First Report of the Colletotrichum siamense species complex causing Anthracnose on Photinia × fraseri Dress in China.

Photinia × fraseri Dress is mainly distributed in the southeast and east of Asia and North America and has been widely cultivated in China. In summer 2018, an anthracnose disease of P. × fraseri Dress was found in a park in Nanjing City, China. Disease leaves showed small, round, light reddish brown spots in the early stage of infection that gradually expanded into round spots, with light gray in centers and brown edges. Fresh lesions were cut into 2-3 mm2 sections, sterilized with 75% ethanol for 30 s, followed by 1% NaClO for 90 s, washed with sterile water 3 times, and placed on potato dextrose agar (PDA) with 0.1 mg/mL ampicillin at 25°C. Colonies of a representative strain "HDSN2-1" were white to greenish grey, and the daily growth rate was 9.5 to10.5 mm/day. Aerial mycelium was grayish white, dense, and cottony, with visible conidial masses at the inoculum point. Conidia were one-celled, smooth-walled, hyaline, with obtuse to rounded ends, with a size of 12.8 to 18.4 × 4.5 to 6.8 µm. Appressoria were one-celled, brown, thick-walled, ellipsoidal, and 7.3 to 10.3 × 5.4 to 6.97µm. The morphological characteristics of HDSN2-1 matched those of the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For further identification, DNA was extracted from HDSN2-1 mycelium and the internal transcribed spacer (ITS) region and partial sequences of β-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1) and calmodulin genes(CAL) were amplified by PCR, and sequenced with primers ITS1/ITS4, TubF1/TubR1, GDF/GDR, CHS-79F/CHS-345R, and CAL1C/CAL2C, respectively(Weir et al. 2012). The sequences were deposited in GenBank [Accession nos: MN889417, MN894596, MN894597, MN894598, MN894599]. A phylogenetic tree was constructed using the maximum likelihood/span>method with concatenated sequences (ITS, TUB2, GAPDH, CHS and CAL) (Zhu et al. 2019). Analyses conducted in MEGA7 placed HDSN2-1 in the C. siamense clade, which includes ex-type ICMP 18578. Pathogenicity of HDSN2-1 was verified on leaves from 7 healthy 8-year-old P. × fraseri and inoculated with either 5-mm mycelial plugs from the edge of 5-day old cultures on PDA or 10 μL of spore suspension (106 conidia/mL),15 healthy plants（8-year-old）were used in 5 repetitions (5 for control, and 10 for the pathogenicity test) in the same way. Controls were treated with PDA plugs or sterile dH2O. The leaves were incubated at 25 ℃ and the inoculated plants were kept in a greenhouse (relative humidity > 85%, 25 ± 1°C). Symptoms were not observed on control plants. Fungal isolates from the symptomatic plants showed the same morphological characteristics with HDSN2-1. C. siamense is a common fungal pathogen of many plants. For example, it was previously reported infecting apples and citrus fruits ( Abirammi et al. 2019). This is the first report of anthracnose of P. × fraseri caused by C. siamense in China.

Colletotrichum gloeosporioides sensu stricto as causal agent of anthracnose on pomegranate fruit in Albania

In spring 2019, an anthracnose-like disease was observed on fruit in pomegranate orchards of cv. Wonderful in the Fier region of Albania. Disease prevalence in the assayed area ranged from 8 to 10%. Symptoms appeared as light to dark brown circular, sunken lesions with increasing diameter, which then merged. This study aimed to characterize the causal agent on the basis of morphology, molecular phylogeny, and pathogenicity. Micro- and macro-morphological features revealed that the disease-associated fungus belonged to Colletotrichum gloeosporioides species complex. A multi-locus molecular phylogeny using partial sequences of barcoding genes/regions (ITS, ACT, CHS-1, GAPDH, TUB2, CAL, GS, and ApMat) ascribed strain CG1 to C. gloeosporioides sensu stricto (s.s.). Pathogenicity tests, performed by inoculating healthy fruit, confirmed C. gloeosporioides s.s. as causal agent of anthracnose on pomegranate fruit in Albania.