Colletotrichum Species Research Articles

Infection of Alfalfa Cotyledons by an Incompatible but Not a Compatible Species of Colletotrichum Induces Formation of Paramural Bodies and Secretion of EVs.

Hemibiotrophic fungi in the genus Colletotrichum employ a biotrophic phase to invade host epidermal cells followed by a necrotrophic phase to spread through neighboring mesophyll and epidermal cells. We used serial block face-scanning electron microscopy (SBF-SEM) to compare subcellular changes that occur in Medicago sativa (alfalfa) cotyledons during infection by Colletotrichum destructivum (compatible on M. sativa) and C. higginsianum (incompatible on M. sativa). Three-dimensional reconstruction of serial images revealed that alfalfa epidermal cells infected with C. destructivum undergo massive cytological changes during the first 60 h following inoculation to accommodate extensive intracellular hyphal growth. Conversely, inoculation with the incompatible species C. higginsianum resulted in no successful penetration events and frequent formation of papilla-like structures and cytoplasmic aggregates beneath attempted fungal penetration sites. Further analysis of the incompatible interaction using focused ion beam-scanning electron microscopy (FIB-SEM) revealed the formation of large multivesicular body-like structures that appeared spherical and were not visible in compatible interactions. These structures often fused with the host plasma membrane, giving rise to paramural bodies that appeared to be releasing extracellular vesicles (EVs). Isolation of EVs from the apoplastic space of alfalfa leaves at 60 h postinoculation showed significantly more vesicles secreted from alfalfa infected with incompatible fungus compared with compatible fungus, which in turn was more than produced by noninfected plants. Thus, the increased frequency of paramural bodies during incompatible interactions correlated with an increase in EV quantity in apoplastic wash fluids. Together, these results suggest that EVs and paramural bodies contribute to immunity during pathogen attack in alfalfa. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A Biological Comparison of Three Colletotrichum Species Associated with Alfalfa Anthracnose in Northern China.

Anthracnose caused by various species of Colletotrichum is one of the most prevalent diseases in alfalfa worldwide that not only reduces forage yields but also severely compromises forage quality. A comprehensive survey was conducted in 2020 in the main production regions of northern China. The survey results showed that alfalfa anthracnose is prevalent in northern China, with the disease incidence ranging from 9% to 45% and the disease index from 5 to 17 (maximum possible score: 100). In total, 24 isolates were collected and identified as three Colletotrichum species (C. trifolii, C. truncatum and C. americae-borealis) based on morphological characteristics and phylogenetic analysis (combined sequences ITS, HIS3, ACT and GAPDH). The three species displayed remarkable environmental adaptability, exhibiting a capacity for growth, sporulation and conidial germination in temperatures ranging from 4 to 35 °C and in different nutrient conditions. Pathogenicity assays showed that C. trifolii was more virulent than the other two species, although the growth vigor (in terms of colony diameter, sporulation and conidial germination) of C. truncatum was the greatest.

Diversity of Colletotrichum Species Assosiated with Imported Citrus Fruits, and their Potential to Infect Chili, Rubber, and Cacao Trees in Indonesia

Spesies Colletotrichum telah banyak dilaporkan sebagai agens penyebab penyakit antraknosa pada tanaman jeruk. Importasi buah jeruk dari beberapa negara produsen ke Indonesia berpotensi membawa masuk spesies Colletotrichum yang belum dilaporkan terdapat di Indonesia. Penelitian dilakukan untuk mengidentifikasi spesies Colletotrichum yang terbawa melalui impor jeruk dan mengevaluasi potensinya menginfeksi tanaman hortikultura dan perkebunan penting di Indonesia. Sebanyak 7 isolat Colletotrichum diisolasi dari buah jeruk impor asal Cina, Pakistan dan jeruk lokal asal Indonesia. Isolat-isolat Colletotrichum tersebut diamati karakter morfologi dan molekulernya serta patogenisitasnya pada tanaman jeruk, cabai, karet, dan kakao. Analisis filogenetik dilakukan dengan metode analisis multilokus gen (multilocus sequence analysis) dengan mengombinasikan lokus gen ITS, ACT, TUB2, dan GAPDH. Identifikasi isolat Colletotrichum menghasilkan satu spesies grup C. boninense kompleks spesies (C. karstii), 3 spesies grup C. gloeosporioides kompleks spesies (C. fructicola, C. gloeosporioides sensu stricto, dan C. siamense). Isolat Colletotrichum asal jeruk Indonesia diidentifikasi sebagai C. gloeosporioides s.s. Strain C. fructicola mampu menginfeksi cabai, sedangkan strain C. karstii menginfeksi cabai, dan kakao. Strain C. gloeosporioides s.s. dan C. siamense memiliki kisaran inang yang lebih luas yaitu cabai, karet, dan kakao. Beberapa spesies Colletotrichum yang terbawa oleh buah jeruk impor berpeluang menjadi patogen pada beberapa tanaman penting di Indonesia, yaitu cabai, karet, dan kakao.

Mechanism of Brevibacillus brevis strain TR-4 against leaf disease of Photinia×fraseri Dress.

Colletotrichum species are among the most common pathogens in agriculture and forestry, and their control is urgently needed. In this study, a total of 68 strains of biocontrol bacteria were isolated and identified from Photinia × fraseri rhizosphere soil. The isolates were identified as Brevibacillus brevis by 16S rRNA. The inhibitory effect of TR-4 on Colletotrichum was confirmed by an in vitro antagonistic experiment. The inhibitory effect of TR-4 was 98% at a concentration of 10 µl/ml bacterial solution, protection of the plant and inhibition of C. siamense was evident. Moreover, the secretion of cellulase and chitosan enzymes in the TR-4 fermentation liquid cultured for three days was 9.07 mol/L and 2.15 µl/mol, respectively. Scanning electron microscopy and transmission electron microscopy confirmed that TR-4 destroyed the cell wall of C. siamense, resulting in leakage of the cell contents, thus weakening the pathogenicity of the bacteria.

Colletotrichum species associated with loquat anthracnose in Kagawa and Tokushima prefectures, Japan

Colletotrichum species associated with loquat anthracnose in Kagawa and Tokushima prefectures, Japan

Synthesis and Evaluation of Copper Oleate Nanoparticles Against Citrus Anthracnose Caused by Colletotrichum gloeosporioides: In Situ Experiments, In Vitro Bioassays, and Field Trials

AbstractAnthracnose in citrus is a severe disease caused by Colletotrichum species. Synthetic fungicides have been used to combat this citrus disease; however, they may have adverse health effects on humans and cause harmful environmental impacts. During a search for environmentally friendly fungicides, we successfully prepared organic copper (copper (II) oleate) nanoparticles by using natural chitosan polymer as a coating agent and stabilizer and by combining an exchange reaction and in situ coating method. Organic copper nanoparticles coated with chitosan polymer (Cu‐oleate@CS NPs) with Cu contents ranged from 5.0 to 6.0 % by weight. The nanoparticles have an average particle size ranging from 50 nm to 60 nm (TEM), covered by a layer of polymer with a thickness of about 10 to 12 nm. The analytical results of zeta potential and dynamic solution size showed that the nanoparticles are uniformly dispersed (90–120 nm by DLS), with high stability (zeta potential=−61.7 mV) and low cytoxicity to Vero healthy cell line. Cu‐oleate@CS NPs significantly inhibited the mycelial growth of C. gloeosporioides in vitro at 500 and 1000 μg/mL. In field trials, the best antifungal efficacy of Cu‐oleate@CS NPs against citrus anthracnose on orange plants was found at a spray concentration of 0.33 % with a disease control efficacy of 54.18 % at 14 days after the second spraying. Our study results demonstrated a novel preparation of Cu‐oleate@CS NPs and suggested that it could be used as a promising nanoformulation to control citrus anthracnose in vitro and in vivo.

Colletotrichum paridis sp. nov., a novel endophytic species on Paris polyphylla var. chinensis

The genus Paris is a crucial medicinal herb, widely cultivated in East Asia and Europe. In 2017 a novel Colletotrichum species was isolated from leaf lesions with grey mould of Paris polyphylla var. chinensis in Sichuan Province of China. Through single-spore technique, four isolates were obtained for characterization. Phylogeny inferred from multi-locus sequences of ITS, gapdh, act, tub2, chs-1, and his3, showed that these isolates represented a novel sister species to C. jinshuiense within the C. dematium complex. Morphologically, this new species produces larger conidia than those of C. jinshuiense and cultures on PDA show a noticeable pink ring. Tests showed that this species had no pathogenicity to the host plants. Based on phylogenetic, morphological, and pathogenicity data, we propose the name C. paridis sp. nov. for this new endophyte from P. polyphylla in China.

Anthracnose disease of Ficus benjamina caused by Colletotrichum orchidearum in Iran

Colletotrichum species cause the anthracnose disease that affects a wide variety of fruit, vegetable, and ornamental hosts. During the summer of 2018–2019, infected leaves of Ficus benjamina var. variegata with typical anthracnose symptoms were collected from several greenhouses in Miyandoab (West Azarbaijan Province) and Maraghe (East Azarbaijan Province) counties, Iran, with an incidence of 10–21%. The fungus was isolated and identified as Colletotrichum orchidearum, based on morphological features and multi-gene sequence analyses (ITS‒rDNA, GAPDH, CHS-1, HIS3, ACT and TUB2 genes). Pathogenicity assays were conducted on two-year-old potted plants with conidial suspensions under greenhouse conditions. Typical anthracnose symptoms including fungal conidiomata were developed on the inoculated leaves, while control plants remained symptomless. Re-isolation and re-identification of the inoculated fungi confirmed Koch’s postulates. This study provides the first evidence of C. orchidearum causing anthracnose disease on Ficus benjamina var. variegata in Iran and the world.

Diversity of Colletotrichum species associated with anthracnose on Euonymus japonicus and their sensitivity to fungicides

Diversity of Colletotrichum species associated with anthracnose on Euonymus japonicus and their sensitivity to fungicides

First Report of Anthracnose on Euonymus japonicus Thunb. Caused by a Provisionally Novel Species of Colletotrichum in the Magnum Complex in South Korea.

Euonymus japonicus Thunb., also known as the evergreen spindle tree, is an evergreen tree, which is widely planted as a hedge plant along streets in South Korea. In April 2022, severe anthracnose symptoms were observed on the leaves of this tree in Jangsu in the Jeonbuk Province of the country (35°43'49.44″N, 127°34'53.7″E). About 80% of the leaves of each affected tree within a 0.03-ha area showed incidence of the disease on approximately 30 trees were planted along the roadside (~30 m). These symptoms typically included circular or irregularly shaped whitish-gray lesions with a diameter of 2.0 to 3.0 cm. In cases where some leaves were severely affected, larger blotches formed. To isolate the pathogen, about ten leaves showing anthracnose symptoms on each tree were randomly selected and brought to the laboratory. Fungal isolations were made from acervuli filled with conidial masses on infected evergreen tissues, followed by plating onto 2% potato dextrose agar (PDA) as well as incubated at 25℃. On the PDA, colonies were circular, raised, green-grey or dark grey, and had a distinct white margin. The conidia were single-celled, transparent, cylindrical with rounded ends, had smooth walls, with a length ranging from 12 μm to 16.7 μm and a width raging from 4 μm to 6.5 μm (av. = 14.1 X 5.0 μm, n=40). Of those that were successfully recovered with approximately 90% frequency, two monoconidial isolates were deposited to the culture collection at Chungnam National University in South Korea (Accession number: CDH059-060). To ensure the identity of the fungus, genomic DNAs were extracted from the selected isolates, CDH059-060, and were sequenced. This was achieved based on partial sequences of the internal transcribed spacer (ITS), actin and beta-tubulin (TUB2) gene regions which were amplified using ITS1F / ITS4 (Gardes and Bruns 1993; White et al. 1990), ACT-512F / ACT-783R (Carbone and Kohn 1999), and T1 / Bt2b (O'Donnell and Cigelnik 1997; Glass and Donaldson 1995) primer pairs, respectively. The resulting sequences were deposited to GenBank (OR984424-425) for ITS, (OR996289-290) for actin, and (OR996291-292) for TUB2. For a phylogenetic analysis, sequences from different gene regions (ITS, actin and TUB2) retrieved from GenBank were aligned, concatenated, and analyzed as a single dataset based on a maximum likelihood analysis. The phylogenetic result revealed that the fungus isolated in this study was positioned in a clearly distinct lineage, provisionally representing an undetermined species of Colletotrichum, which is most closely related to Colletotrichum liaoningense (Y.Z. Diao, C. Zhang, L. Cai & X.L. Liu, CGMCC3.17616 (KP890104 for ITS, KP890097 for actin, and KP890111 for TUB, Diao et al. 2017). Sequence comparisons revealed that this pathogen differed from C. liaoningense at 20 of 494 characters (∼4.0%) in the ITS and 2 of 251 (∼1.0%) in the actin sequences. For pathogenicity tests, three seedlings of E. japonicus were used. The leaves for each tree were treated with 10 ml of a conidial suspension by spraying (1x10⁶ conidia ml-1 of the isolate, CDH059), while the three seedlings were treated with distilled water as control. After sprayed, the treated areas were sealed with plastic bags for a day to maintain humidity. Anthracnose symptoms identical to those observed in the field appeared seven days after inoculations, while no symptoms were observed in the control. Re-isolations were successfully achieved from the treatments, fulfilling Koch's postulates. Anthracnose associated with the provisionally novel species of Colletotrichum sp. on E. japonicus has not been recorded elsewhere, and in this regard, this is the first report of anthracnose caused by Colletotrichum sp. on E. japonicus in Korea. To effectively control the disease, more attention should be paid to the host range of the pathogen and other regions where the disease caused by the pathogen might occur in the country.

Identifying and Controlling Anthracnose Caused by Colletotrichum Taxa of Welsh Onion in Sanxing, Taiwan.

Leaves of Welsh onion (Allium fistulosum) are subject to various fungal diseases such as anthracnose (Colletotrichum species) and Stemphylium leaf blight (Stemphylium vesicarium). These diseases are the main biotic limitations to Welsh onion production in northern Taiwan. From 2018 to 2020, anthracnose symptoms were observed throughout Welsh onion fields in northern Taiwan, mainly the Sanxing area. In total, 33 strains of Colletotrichum species were isolated from diseased leaves, and major causative agents were identified based on a multilocus phylogenetic analysis using four genomic regions (act, tub2, gapdh, and internal transcribed spacer). Based on this phylogeny, Colletotrichum species causing anthracnose of Welsh onion were identified as C. spaethianum (C. spaethianum species complex) and C. circinans (C. dematium species complex) in the Sanxing area, northern Taiwan. To determine and compare the pathogenicity of each species, representative fungal strains of each species were inoculated on the cultivar 'Siao-Lyu' by spraying spore suspension onto the leaf surface. Welsh onion plants were susceptible to both species, but disease incidence and severity were higher in C. spaethianum. In total, 31 fungicides were tested to determine their efficacy in reducing mycelial growth and conidial germination of representative strains of C. spaethianum and C. circinans under laboratory conditions. Five fungicides-fluazinam, metiram, mancozeb, thiram, and dithianon-effectively reduced mycelial growth and spore germination in both C. spaethianum and C. circinans. In contrast, difenoconazole and trifloxystrobin + tebuconazole, which are commonly used in Welsh onion production in northern Taiwan, mainly the Sanxing area, were ineffective. These results serve as valuable insights for growers, enabling them to identify and address the emergence of anthracnose caused by C. spaethianum and C. circinans of Welsh onion, employing fungicides with diverse modes of action. The findings of this study support sustainable management of anthracnose in Sanxing, northern Taiwan, although further field tests of the fungicides are warranted.

Colletotrichum species associated with atemoya anthracnose in northeast Brazil

Anthracnose, caused by Colletotrichum spp., is an important disease of atemoya that can infect and cause damage to different plant organs. The symptoms include foliar anthracnose, flower abortion, branch tip bleaching, and necrotic lesions on fruits. Knowing the etiology of this disease is essential for properly developing management strategies. Therefore, this study aimed to identify Colletotrichum species associated with atemoya in the Northeast region of Brazil, based on multi-locus phylogeny and morphological analyses, to better understand the etiology of anthracnose in this plant species. The Colletotrichum isolates were obtained from symptomatic leaves and branches of atemoya collected in commercial fields in Vitória da Conquista (Bahia), Palmeira dos Índios and Rio Largo (Alagoas), located in northeast Brazil. The initial analysis of the partial sequences of the GAPDH gene was performed with nineteen Colletotrichum isolates obtained from leaves and branches of atemoya trees with typical anthracnose symptoms. Seventeen isolates were grouped in the gloeosporioides complex and two in the boninense complex. Therefore, eleven isolates were submitted to concatenated phylogenetic analyses with the genes GAPDH, TUB2, and ITS region of the rDNA to confirm the identity of the isolates at the species level. The species C. theobromicola, C. siamense, C. fructicola and C. karstii were identified and are associated with atemoya anthracnose in the Northeast region of Brazil.

Suppression of anthracnose disease by orsellinaldehyde isolated from the mushroom Coprinus comatus.

Anthracnose caused by Colletotrichum species is one of the most devastating diseases of fruits and crops. We isolated and identified an antifungal compound from the mushroom Coprinus comatus and investigated its inhibitory potential against anthracnose disease-causing fungi with the goal of discovering natural products that can suppress anthracnose-caused plant disease. The culture filtrate of C. comatus was subjected to a bioassay-guided isolation of antifungal compounds. The active compound was identified as orsellinaldehyde (2,4-dihydroxy-6-methylbenzaldehyde) based on mass spectroscopy and nuclear magnetic resonance analyses. Orsellinaldehyde displayed broad-spectrum inhibitory activity against different plant pathogenic fungi. Among the tested Colletotrichum species, it exhibited the lowest IC50 values on conidial germination and germ tube elongation of Colletotrichum orbiculare. The compound also showed remarkable inhibitory activity against Colletotrichum gloeosporiodes. The staining of Colletotrichum conidia with fluorescein diacetate and propidium iodide demonstrated that the compound is fungicidal. The postharvest in-vivo detached fruit assay indicated that orsellinaldehyde suppressed anthracnose lesion symptoms on mango and cucumber fruits caused by C. gloeosporioides and C. orbiculare, respectively. Orsellinaldehyde was identified as a potent antifungal compound from the culture filtrate of C. comatus. The inhibitory and fungicidal activities of orsellinaldehyde against different Colletotrichum species indicate its potential as a fungicide for protecting various fruits against anthracnose disease-causing fungi.

Essential Oils for Managing Anthracnose in Mango (Mangifera indica): Laboratory Results Do Not Translate into Field Efficacy.

Essential oil-based products with broad plant disease control claims are commercially available and may be a practical alternative to copper fungicides for crop protection in organic mango orchards. We evaluated the disease control efficacy and crop safety of thyme oil, savory oil, and tree tea oil through replicated in vitro, in vivo (detached leaf and potted trees), and field assays. Three Colletotrichum species associated with mango anthracnose were tested in vitro, whereas only C. siamense was used for in vivo assays. Within the range of concentrations tested in vitro (62.5 to 2,000 μl active ingredient [a.i.]/liter), thyme and savory oil displayed fungicidal activity, whereas no fungistatic or fungicidal activity was observed with tea tree oil. In the in vivo assays, none of the treatments based on a preventive application rate of thyme (1,150 μl a.i./liter), savory (2,000 μl a.i./liter), or tea tree oil (342 μl a.i./liter) were effective in preventing the development of anthracnose on wounded and artificially inoculated leaves. Although field applications of thyme or tea tree oil did not result in phytotoxicity or negative impacts on fruit yield, they were ineffective in reducing the incidence and severity of naturally occurring anthracnose. Applications of copper hydroxide approved for organic agriculture were effective in controlling anthracnose in the field, and no added benefits were found by premixing this compound with thyme oil. Results indicate that essential oil products based on thyme or tea tree oil are inefficient at controlling anthracnose in mangoes.

Diversity, Prevalence and Virulence of Colletotrichum Species Causing Anthracnose on Cassava Leaves in the Northern Region of Brazil.

Cassava (Manihot esculenta Crantz) is a staple crop widely cultivated by small farmers in tropical countries. However, despite the low level of technology required for its management, it can be affected by several diseases, with anthracnose as the main threat. There is little information about the main species of Colletotrichum that infect cassava in Brazil. Thus, the objective of this work was to study the diversity, prevalence and virulence of Colletotrichum species that cause anthracnose in cassava leaves in northern Brazil. Twenty municipalities of the Pará and Tocantins states were selected, and leaves with symptoms were collected in those locations. Pure cultures were isolated in the laboratory. Species were identified using phylogenetic analyses of multiple loci, and their pathogenicity, aggressivity and virulence levels were assessed. Our results showed the greatest diversity of Colletotrichum associated with anthracnose in cassava plants of the "Formosa" cultivar in the Tocantins and Pará states. We determined the presence of Colletotrichum chrysophilum, C. truncatum, C. siamense, C. fructicola, C. plurivorum, C. musicola and C. karsti, with C. chrysophilum as the most aggressive and virulent. Our findings provide accurate identifications of species of Colletotrichum causing anthracnose in cassava crops, which are of great relevance for cassava breeding programs (e.g., the search for genotypes with polygenic resistance since the pathogen is so diverse) and for developing anthracnose management strategies that can work efficiently against species complexes of Colletotrichum.

First Report of Anthracnose Caused by Colletotrichum siamense on Machilus thunbergii Siebold & Zucc. in South Korea.

Machilus thunbergii Siebold & Zucc., known as Japanese bay tree, is an evergreen tree distributed widely in East Asia, including South Korea, where the species is of ecological importance. Machilus thunbergii provides habitat for wildlife species and is a common urban tree. In September 2022, anthracnose symptoms on leaves were observed in Jeju (33°26'02.4"N, 126°19'48.8"E) and Tongyeong (34°49'27.1"N, 128°24'01.8"E) in South Korea. Disease incidence on leaves of each affected tree, naturally growing in an urban forest area covering approximately 0.5 ha was approximately ~ 70 % in each study area. Anthracnose symptoms that were observed on 70 to 80% leaves per tree in each study area included orbicular or irregular, whitish-grey spots on leaves that were 1.5 to 3.0 cm in diam. In some cases where leaves were severely affected, larger blotches were formed, leading to bleaching symptoms and eventually defoliation. For pathogen isolation, two or three leaves showing anthracnose symptoms from each of the 15 trees were randomly selected and brought to the laboratory. Fungal isolations were then directly made by transferring spores from acervuli that developed on diseased leaves onto potato dextrose agar (PDA) media. Cushion shaped acervuli filled with salmon to orange-colored conidial masses were produced on media approximately two weeks after the incubation at 25 ± 1°C with a photoperiod of 12 h. Conidia were single celled, hyaline, cylindrical with rounded ends, smooth walls, 13.7 to 18.1 μm long and 3.1 to 4.5 μm wide (n=30). Among 15 cultures that were successfully isolated, 10 isolates were retained based on culture characteristics, and two randomly selected monoconidial cultures were deposited in the culture collection (CDH) of the Chungnam National University, Republic of Korea (Accession No. CDH057-58). Two isolates selected, CDH057 and CDH058, were subjected to identification, and this was achieved based on multiplesequence comparisons using on internal transcribed spacer regions of rDNA (ITS1 and ITS2), partial sequences of actin (ACT) and β-tubulin (TUB2) gene regions amplified using ITS1F / ITS4, ACT-512F / ACT-783R and T1 / Bt2b, respectively (Weir et al. 2012). The representative sequence data were deposited in GenBank under the accession numbers OR473277 and OR473278 for the ITS, OR480772 and OR480773 for ACT, and OR480774 and OR480775 for TUB2. The resulting sequences were further used for a phylogenetic analysis based on the maximum likelihood method using a concatenated dataset of the ITS, ACT and TUB2 gene sequences for Colletotrichum species in the C. gloeosporioides clade. The results showed that the pathogen isolated in this study clustered with Colletotrichum siamense (Vouchered specimens: MFLU 090230, COUFPI291, and COUFPI294) (Prihastuti et al. 2009). Sequence comparisons revealed that the isolates obtained in this study differed from the type species of C. siamense (MFLU 090230; FJ972613 for ITS, FJ 907423 for ACT, FJ907438 for TUB2) at 2 of 258 bp (∼0.8%) and 6 of 387 bp (∼1.6%) in the ACT and TUB2 sequences, respectively, while the ITS was identical to the type species. For pathogenicity tests, a total of ten three-year-old seedlings of M. thunbergii were used. The leaves of each tree were sprayed with 5 ml of conidial suspension (105 conidia/ml, isolate CDH057). Three control plants were sprayed with sterile water. After being sprayed, treated areas were sealed with a plastic bag for 24 hours to preserve humidity. Anthracnose symptoms, identical to those observed in the field, appeared five to seven days after the inoculations, while no symptoms were observed on control plants. The isolates used in the pathogenicity test were reisolated from 90% of lesions, and their identity was confirmed based on sequence comparisons, thus fulfilling Koch's postulates. Species of the C. gloeosporioides species complex include important plant pathogens, particularly C. siamense, which cause significant losses of economic and ecological relevance on a wide range of hosts (~ 100 hosts) (Talhinhas and Baroncelli 2021). Although C. fioriniae in the C. acutatum species complex, was found on M. thunbergii in South Korea (Thao et al. 2023), anthracnose associated with C. siamense on M. thunbergii has not been reported in the country. In this regard, this is the first report of anthracnose caused by C. siamense on M. thunbergii in South Korea. To effectively control the disease, more attention should be paid on the host range of the pathogen and other regions where the disease caused by the pathogen might occur in the country.

Apple Bitter Rot and Glomerella Leaf Spot: A Comprehensive Review of Causal Species and Their Biology, Fungicide Sensitivities, and Management Strategies.

Bitter rot and Glomerella leaf spot (GLS) are two distinct diseases of apple fruit and foliage caused by members of the ascomycete fungal genus Colletotrichum. While GLS is restricted to subtropical and in some areas to temperate climates, bitter rot is responsible for significant yield loss worldwide, particularly during the post-harvest period. Initially thought to be caused by just two species of Colletotrichum, C. acutatum and C. gloeosporioides, advances in molecular biology and sequencing techniques enabled the identification of 25 different species capable of causing bitter rot and/or GLS of apple belongs to the C. gloeosporioides species complex (CGSC), C. acutatum species complex (CASC) and C. boninense species complex (CBSC). Three species (C. gloeosporioides, C. fructicola, and C. chrysophilum) of CGSC cause both bitter rot and GLS, 18 species (6 of CGSC and 12 of CASC) only cause bitter rot, and four species (C. aenigma and C. asianum of CGSC, C. limetticola of CASC and C. karstii of CBSC) only cause GLS. These species were found to differ in their geographical distribution, environmental and host tissue preference, pathogenicity, and fungicide sensitivities. In this review, we summarize the distribution, life cycle, and pathogenicity mechanisms of all currently known Colletotrichum species responsible for bitter rot and GLS of apple. Furthermore, we describe known apple defense mechanisms and management strategies for the control of these economically significant pathogens and identify gaps in our present understanding for future research.

First report of anthracnose of Lithocarpus polystachyus caused by Colletotrichum fructicola in China.

Lithocarpus polystachyus (Wall. ex A. DC.), an economically valuable plant species belonging to the Fagaceae family, has been used as herbal tea to prevent diabetes because of the high content of flavonoids and dihydrochalcones in the leaves (Shang et al. 2022). In July 2022, the severe leaf lesion on L. polystachyus was first observed in Yongshun County, Xiangxi autonomous prefecture (28°45'34''N, 109°40'11''E), Hunan province, China. Yongshun County is characterized by hills and mountains, situated in a subtropical region with a mild and humid climate. A second outbreak in July 2023 was observed in the same area. The observed incident rates in the past two years were 87.3% and 90.6%, respectively. Once infected, almost all plant leaves will be infected, leading to a substantial reduction in the yield of L. polystachyus. The disease presented symptoms characterized by round or irregularly shaped lesions that initially manifested as brown spots. These lesions frequently merged into larger, dark-brown areas along the leaf margins before eventually wilting. To ascertain the pathogenic species responsible for this disease, fungal isolation was conducted using a tissue separation method (Xu et al. 2023). The infected leaf tissues were surface-disinfected with 75% ethanol and 0.1% HgCl then small pieces (1×1 cm), were placed onto potato dextrose agar (PDA) medium (Sigma-Aldrich, 70139) and incubated at 28°C for 6-9 days. Colonies were villiform and initially white, becoming gray after 6 days. Sterilized dissecting needles were used to pick single hyphal tips from the edge of the colonies and placed onto PDA for strain purification. After 15 days, the purified colonies grew fluffy white hyphae with abundant conidia. The conidia were cylindrical, had round ends, and ranged from 5.75 to 14.83 μm long and 1.75 to 2.38 μm wide (n=50). According to morphological and cultural characteristics, these isolates were preliminarily identified as Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde (Damm et al. 2012). To further affirm the identity of the pathogen, DNA was extracted from mycelia using a DNA extraction kit (Sigma-Aldrich, G2N70). The internal transcribed spacer (ITS) region, the transcription elongation factor (TEF), and the actin (ACT) gene were then amplified from genomic DNA extracted from three isolates (Cof1, Cof2, and Cof3) using specific primers. The primers utilized were ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R and ACT-512F/ACT-783R (Carbone and Kohn 1999) for ITS region, transcription elongation factor gene and actin gene amplification, respectively. Sequence identity indicated that these isolates were highly homologous to C. fructicola. The ITS (Genbank No. PP002156, OR880553 and OR880554), TEF (No. PP061421, PP061422 and PP061423), and ACT (No. PP061418, PP061419 and PP061420) sequences of the isolates Cof1, Cof2, and Cof3 shared 99 to 100% identity with their counterparts (No. OR083309, MF627961, and OQ427895) in C. fructicola, respectively. A neighbor-joining phylogenetic tree constructed using MEGA11 (Tamura et al. 2021) also indicated that these isolates were C. fructicola. Both morphological and molecular characteristics confirmed the identification of this pathogen as C. fructicola. Colletotrichum species are known to cause anthracnose disease in a variety of economically important crops (Sharma and Kulshrestha 2015). To further validate the ability of the isolated C. fructicola to induce the same symptoms as observed in the field, the pathogenicity assay was assessed following Koch's postulates (Gradmann, 2014). Conidial suspensions (1×105 conidia per mL) from three isolates were individually inoculated onto artificially wounded leaves of 3-year-old L. polystachyus. Negative controls were established by inoculating leaf wounds with sterile distilled water. The plants were incubated in a greenhouse at 28°C and 90% humidity with a 12-h photoperiod. The experiment was replicated three times. Necrotic lesions were observed on all pathogen-inoculated wounds within 6 days after inoculation, whereas controls showed no observable symptoms. Morphological and molecular characterization of re-isolated pathogens from infected leaves indicated that the pathogens were identical. To our knowledge, this is the first report of anthracnose of L. polystachyus caused by C. fructicola in China. Farmers in the local mountainous areas are economically reliant on L. polystachyus production, while anthracnose has caused over half of the trees to lose their commercial value, resulting in significant economic losses. Our findings hold great promise for advancing strategies in the prevention and treatment of anthracnose in L. polystachyus.

In vitro Evaluation of Fungicides against Anthracnose of Betelvine (Piper betle L.)

Background: Betelvine is important commercial crop and the most profitable among all cultivated crops, which plays a vital role in the overall livelihood security of farm families. Diseases are the major yield constraints of crop plants. One of the most serious fungal diseases of dragon fruit is anthracnose caused by Colletotrichum species. Since less information available on anthracnose of betel vine, this study was undertaken. Methods: The efficacy of non-systemic, systemic and combination fungicides were tested against Colletotrichum gloeosporioides using poisoned food technique (Vincent 1947) under in vitro condition. Six non-systemic fungicides Chlorothalonil 75% WP, Captan 50% WP, Mancozeb 75% WP, Copper oxychloride 50% WP, Propineb 70% WP and Copper hydroxide 53.8% at (250 ppm, 500 ppm and 1000 ppm), six systemic fungicides Hexaconazole 5% EC, Propiconazole 25 % EC, Azoxystrobin 25% SC, Tebuconazole 25.9% EC, Difenaconazole 25% EC and Picoxystrobin 22.5% SC at (100ppm, 150ppm, 250ppm) and six combi fungicides Propiconazole 13.9% + Difenoconazole 13.9% EC, Tebuconazole 50% + Trifloxystrobin 25% WG, Fluropyram 200 g/L + Tebuconazole 200 g/L SC, Fluxopyroxad 250 g/l + pyraclostrobin 250g/L, Fluopyram 250 g/L + Trifloxystrobin 250 g/L SC, Azoxystrobin 16.7% + Tricyclazole 33.3% SC at (150 ppm, 250 ppm, 500 ppm) were evaluated. Results: Among six non-systemic fungicides evaluated against C. gloeosporioides which was obtained from the isolated sample and results revealed that the Copper hydroxide gave 69.90 % inhibition which was superior over all other fungicides evaluated and least inhibition was recorded with Mancozeb 40.30%. Difenconazole, Tebuconazole were the best systemic fungicides found best with inhibition % of 98.28 and 95.17 when evaluated against C. gloeosporioides. Out of the six evaluated combination products, propiconazole + difenconazole exhibited the highest inhibition rate at 99.78 %. Following closely, Fluopyram 200 g/L + Tebuconazole 200 g/L SC and Tebuconazole 50% EC + Trifloxystrobin 25% WG displayed inhibition rates of 89.47% and 87.50 % respectively.

Identification and Characterization of Colletotrichum Species Associated with Anthracnose Disease of Plum.

Plum (Prunus salicina Lindl.) is commercially cultivated worldwide for the high levels of nutrients in the fruit. In recent years, anthracnose has been severe in some plum planting areas in China, resulting in a large number of necrotic leaves, blight, and premature leaf fall. In this study, anthracnose samples of plum leaves were collected from Hezhou, Guilin, and Lipu in Guangxi Province and Meishan, Abe Tibetan, and Qiang Autonomous Prefecture of Sichuan Province. Characteristics of mycelia on potato dextrose agar, morphology of appressoria and conidia, and analysis of sequences of several marker regions (internal transcribed spacer [ITS] region, glyceraldehyde-3-phosphate dehydrogenase [GAPDH], chitin synthase [CHS-1], histone H3 [HIS3], actin [ACT], β-tubulin [TUB2], and the intergenic region between apn2 and MAT1-2-1 [ApMat]). The resulting 101 Colletotrichum isolates obtained were identified as eight species: C. fructicola (50.5%), C. siamense (24.8%), C. karsti (8.9%), C. plurivorum (7.9%), C. aeschynomenes (3.9%), C. gloeosporioides (2%), C. celtidis (1%), and C. phyllanthi (1%). Representatives of all eight Colletotrichum species were found to cause disease on wounded leaves of plum seedlings in pathogenicity assays. As far as we are aware, this is the first report of anthracnose of plum caused by C. celtidis and C. phyllanthi in China.