Anthracnose Symptoms Research Articles

First report of Colletotrichum gloeosporioides causing anthracnose on grapevine (Vitis vinifera) in Shaanxi province, China.

Grape anthracnose caused by Colletotrichum spp. is an economically important disease in most vineyards during rainy and humid weather. In June 2021, widespread anthracnose was observed in leaves of Vitis vinifera cv. Red Globe in Yangling city (latitude = 34.28, longitude = 108.07), Shaanxi Province. Diseased plants exhibited circular brown necrotic lesions with yellow borders in young leaves (Fig. 1A). Approximately 80% of the 75 grape plants showed disease symptoms that were consistent with those previously reported for anthracnose on grape caused by C. acutatum, C. gloeosporioides, and C. viniferum (Hong et al. 2008; Oo and Oh 2017). To isolate the pathogen, ten diseased leaves were collected. Small pieces (0.5 mm × 0.5 mm) of symptomatic tissue were surface sterilized with 75% alcohol for 30 s, transferred to 4% NaClO for 2 min, and then washed twice with sterile distilled water. The pathogen was isolated and cultured on potato dextrose agar (PDA) at 25 °C with a 12 h light/dark photoperiod. Hyphal tips of the isolate were transferred to fresh PDA after 1 day. Acervuli were formed after 15 days on PDA. The colonies were single-spored to obtain pure cultures. Pure cultures on PDA appear white to grey, and the reverse of the colonies was olive gray or yellowish white (Fig. 1B, C). Conidia were single-celled, hyaline, straight cylindrical with ends rounded, and measured 12.50 to 17.78 μm (length) (mean=15.19 μm, n=50) × 3.66 to 5.82 μm (width) (mean=4.48 μm, n=50) (Fig. 1D). The morphological characteristics matched the previous descriptions of C. gloeosporioides by Sawant et al. (2012). For molecular identification, genomic DNA was extracted from isolate YLRG13, and the actin (ACT), internal transcribed spacer (ITS), and beta-tubulin (TUB2) were amplified using the primer pairs ACT-512F/ACT-783R (Carbone and Kohn 1999), ITS1F/ITS4 (White et al. 1990), and T1/T2 (O' Donnell and Cigelnik 1997), respectively. The ACT (OQ031183 and MZ686707), ITS (OP999326 and MZ669974), and TUB2 (OQ031184 and MZ686708) nucleotide sequences of isolates YLRG5 and YLRG13 were deposited in GenBank. BLASTn showed 99 to 100% similarity with C. gloeosporioides. Neighbor-joining trees based on the three genes was constructed using PAUP version 4.0b10. The results showed the isolated YLRG5 and YLRG13 from V. vinifera was closely related to C. gloeosporioides with high bootstrap values (Fig. S2). Pathogenicity testing was performed with isolate YLRG13 by 10 μL conidial suspension (4×108 conidia/ml) on the surfaces of five wounded detached leaves of six-year-old Vitis vinifera cv. Red Globe. Sterile distilled water was used as a control. Inoculated plants were placed in a humid chamber at 25 °C. Conidial germination was observed at 24 hours post-inoculation (Fig. S1E). Four days after inoculation, typical anthracnose symptoms developed on inoculated grape leaves (Fig. S1F). No symptoms were observed on the controls. The fungus reisolated from the symptomatic grape leaves showed the same morphological characteristics of the inoculated isolate, fulfilling Koch's postulates. To our knowledge, this is the first report of grape anthracnose caused by C. gloeosporioides in Northwestern of China. C. gloeosporioides was reported causing anthracnose on a wide variety of horticultural crops, such as almond, avocado, apple, banana, cashew, citrus, cucumber, grape, guava, mango, onion, papaya, passion fruit, pepper, strawberry, tomato, and watermelon (Sharma and Kulshrestha 2015), suggesting the potential cross-infection between grape and other horticultural crops.

First report of Colletotrichum fioriniae causing anthracnose on pear fruit in Serbia.

European pear (Pyrus communis L.) is one of the most important fruits in Serbia, with a total production of 55,938 tonnes in 2021 (Statistical Office of the Republic of Serbia). In November 2021, pear fruits (cv. Santa Maria) with typical anthracnose symptoms were collected in one storage facility in the area of Smederevo, Serbia. The disease incidence was about 2%. A total of nine infected fruits had dark, circular, sunken necrotic lesions with concentric rings of orange conidial masses produced in black acervuli. The tissue pieces from the edges of lesions (3 to 5 mm2) were surface-sterilized in 1% sodium hypochlorite for 1 min, rinsed twice in sterilized distilled water, air-dried, and incubated on potato dextrose agar (PDA) at 25°C. One isolate/diseased fruit was obtained (nine in total). Colony morphology of all isolates on PDA was light gray to pinkish on the upper side, and pink to vinaceous on reverse after 7 days of incubation at 25°C in the dark. Average growth rate was 64 ± 2 mm. The conidia were hyaline, fusiform, aseptate, smooth-walled, with both ends acute, and measured 12.8 to 16.6 µm × 3.5 to 4.5 µm (n =100). Appressoria were single, smooth-walled, dark brown, clavate to irregular outline, 5.5 to 9.6 × 4.8 to 7.2 μm (n =20). These morphological characterstics are consistent with the description of Damm et al. (2012) for Colletotrichum fioriniae. To confirm this identification, genomic DNA of representative isolate SZ-21-36 was extracted and the ITS region of the rDNA, partial beta-tubulin gene (BenA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and partial actin gene (ACT) were amplified using the primers ITS1/ITS4, Bt2a/Bt2b, GDF1/GDR1, and ACT512F/ACT783R, respectively (Hassan et al. 2019). The nucleotide sequence of each locus has been deposited in GenBank under Accession Nos. ON171625 (ITS), ON186696 (BenA), ON186697 (GAPDH), and ON186698 (ACT). Multilocus phylogenetic analysis based on the above mentioned molecular markers placed Serbian isolate of C. fioriniae with other isolates of this species deposited in GenBank (bootstrap support of 100%). Pathogenicity test was conducted on symptomless, detached pear fruits (cv. Santa Maria). Five surface-sterilized fruits were wound-inoculated with 50 μl of a conidial suspension (1 × 106 conidia/ml). After 10 days of incubation in a plastic chamber (25°C, 90% relative humidity, and 12-h photoperiod), typical anthracnose lesions developed on inoculated fruits. Five control fruits inoculated with sterile distilled water remained asymptomatic. The experiment was repeated once. Fungal colonies reisolated from the lesions showed the same morphological features as the original isolate. Pathogen was not reisolated from the control fruits. C. fioriniae was previously reported on European pear fruits in Croatia (Ivić et al. 2013), France (Da Lio et al. 2017), China (Fu et el. 2019), and in the USA (Pavlović et al. 2019). To our knowledge, this is the first report of anthracnose of pear fruit caused by C. fioriniae in Serbia. Currently, the economic impact of anthracnose caused by this pathogen is minimal, but its presence decreases commercial value and quality of pear fruits, and can be a risk to the further spread of C. fioriniae to other plant species.

Anthracnose Controlled by Essential Oils: Are Nanoemulsion-Based Films and Coatings a Viable and Efficient Technology for Tropical Fruit Preservation?

Post-harvest diseases can be a huge problem for the tropical fruit sector. These fruits are generally consumed in natura; thus, their integrity and appearance directly affect commercialization and consumer desire. Anthracnose is caused by fungi of the genus Colletotrichum and affects tropical fruits, resulting in lesions that impair their appearance and consumption. Antifungals generally used to treat anthracnose can be harmful to human health, as well as to the environment. Therefore, essential oils (EO) have been investigated as natural biofungicides, successfully controlling anthracnose symptoms. The hydrophobicity, high volatility, and oxidative instability of essential oils limit their direct application; hence, these oils must be stabilized before food application. Distinct delivery systems have already been proposed to protect/stabilize EOs, and nanotechnology has recently reshaped the food application limits of EOs. This review presents robust data regarding nanotechnology application and EO antifungal properties, providing new perspectives to further improve the results already achieved in the treatment of anthracnose. Additionally, it evaluates the current scenario involving the application of EO directly or incorporated in films and coatings for anthracnose treatment in tropical fruits, which is of great importance, especially for those fruits intended for exportation that may have a prolonged shelf life.

Etiology of Colletotrichum diseases on Sastuma mandarin in Croatia

During the last decade anthracnose has become a major disease of Satsuma mandarin, the most important citrus crop in Croatia. The aim of this study was to determine Colletotrichum species associated with different symptoms and to identify the origin of inoculum. From 2013 to 2016, 437 samples of plant material were collected. Colletotrichum spp. was isolated from 93% of dried twigs, 35% of dropped flowers, 89% of leaf spots, all fruit (100%) with anthracnose or calyx-end rot symptoms, 12% of fruit with post-harvest soft rot and from 40% of fruit showing spots remaining on trees after harvest. Out of 258 Colletotrichum isolates, 253 has been morphologically identified as C. gloeosporioides (Penz.) Penz. & Sacc. species complex. Twenty-seven representative isolates were selected for phylogenetic analysis. Sequencing the inter-spacer gene region of ribosomal DNA confirmed the identity of the species. Artificial inoculation of flowers led to more than 2-fold higher young fruit drop compared to control. Pathogenicity tests on green fruit induced typical anthracnose symptoms on 82% of inoculated fruit two months after inoculation. Inoculation of mature fruit caused the appearance of typical anthracnose symptoms on 87% of inoculated fruit. These results showed that C. gloeosporioides species complex is responsible for different disease types on Satsuma mandarin, and that the fungus is present throughout the year on different plant organs.

First Report of Colletotrichum fioriniae Causing Grapevine Anthracnose in New York.

Grapevine is one of the most widely-planted fruit crops in the world, and is the most economically important fruit crop in the state of New York, USA. Symptoms of anthracnose on grapevine are similarly widely-reported on grapevine fruit and foliage, and such symptoms are commonly attributed to Elsinöe ampelina (Wilcox et al., 2015). However, similar symptoms, if not identical, to those associated with E. ampelina have been sporadically attributed to various species in the genus Colletotrichum. In September 2021, a survey was conducted in three research vineyards at Cornell AgriTech in Geneva, NY. Symptoms of anthracnose werebserved on four Vitis interspecific hybrid breeding lines in a 1 ha vineyard. Leaves, fruit, and petioles showing symptoms of anthracnose, i.e., sunken necrotic lesions with grayish centers and brownish margins, were collected. Symptomatic and healthy portions of surface-sterilized tissues were placed on PDA medium and incubated at 23oC for 7 days. Several petiole samples yielded colonies of white to greyish mycelium, with some red to orange pigmentation (Fig. 1A and 1B), similar to those described by Chowdappa et al. (2009) for Colletotrichum species isolated from grapevine in India. Cultures were allowed to sporulate. Slides from cultures were prepared and examined at 400X magnification. Conidia from cultures were cylindrical with rounded ends, 13.5-15.2 μm in length and 7.6-9.0 μm in width (Fig. 1C). Koch's postulates were fulfilled by inoculating detached healthy leaves of V. vinifera 'Chardonnay' that had been surface sterilized in 10% sodium hypochlorite and triple-rinsed in sterile distilled water. Drop inoculation was used from a suspension of 105 conidia/ml from the foregoing pure cultures as five 2 µL droplets per leaf. Inoculated detached leaves were maintained on water agar in a Petri dish at 23oC. Four days after inoculation, symptoms were observed and compared with the originally collected samples. Inoculated leaves displayed symptoms typically found on the collected tissues, and the original pathogen, as confirmed by colony morphology and conidial characteristics and dimensions, was reisolated from inoculated leaves, and not from non-inoculated controls. For molecular characterization, fungal DNA was isolated by using Qiagen DNeasy kit and amplified using the following primer pairs: ITS1/ITS4, TEF (Hyun et al., 2009), E. ampelina F/R (Santos et al. 2018), TUB2, ACT, HIS3, GAPDH and CHS1 (Damm et al., 2001). PCR products were purified using ExoSAP-IT, and samples were Sanger sequenced. Sequences were analyzed using Geneious Prime software, and the resulting sequences (NCBI accessions OL720215, OL720216, OL720217, OL720218, OL853836, OM982612, OM982613, OM982614, OM982615 and OM982616) had 94 to 100% identity to Colletotrichum fioriniae NCBI accessions MN944922.1, MK646015.1, MN944922.1, MN856415.1, KU847413.1, MN520490.1, MN544294.1, KY695259.1, MN535117.1 and MN544295.1. Symptoms of grapevine anthracnose caused by Colletotrichum species have been reported from India (Chowdappa et al., 2009) and Korea (Kim et al., 2021). To our knowledge this is the first report of grapevine anthracnose caused by C. fioriniae Anthracnose and ripe rot are diseases of increasing importance, particularly as new grapevine cultivars with resistance to powdery mildew or downy mildew are adopted. Taxonomy of the causal agents (E. ampelina and Colletotrichum spp.) has undergone considerable revision. Consequently, distribution and relative prevalence of the various taxa will require further study.

First Report of Anthracnose on Juglans regia Caused by Colletotrichum siamense in Korea.

Walnut (Juglans regia) is one of the main tree crops cultivated for nut production in South Korea with an estimated production of about 1,189 tons per year (Korea Forest Service 2020). In August 2021, anthracnose symptoms, including dark, depressed, irregularly shaped lesions on fruits and leaves of walnut cv. Sinlyeong, were observed at three orchards in Nonsan (36°10'22.5"N 127°06'14.8"E) and Suwon (37°16'04.7"N 126°55'22.3"E and 37°15'10.6"N 126°57'35.6"E). This led to severe yield loss of walnut fruit with a disease incidence of approximately 70 to 80% in each orchard. Three samples, including infected fruits and leaves, were randomly collected per site. Fungal isolates were isolated either from acervuli filled with conidial masses on infected walnut tissues or from plant tissues that were surface-disinfested, followed by plating onto 2% PDA. Colonies were initially white, later became pale brownish to light gray with concentric rings of salmon sporodochia. White to gray aerial mycelia, reaching 65 mm diameter in 5 days, were abundantly produced on PDA at 25 °C. Appressoria were brown, ovoid, and in some cases, clavate, 5.1-8.7 μm in length, and 3.2-5.1 μm in width (n = 50). Conidia were single celled, hyaline, cylindrical with rounded ends and smooth walls, guttulate, 13.6-18.8 µm in length, and 4.4-6.3 µm in width (n = 50). Setae were absent. Three isolates, i.e., one per orchard, were retained and deposited in the culture collection (CDH) of National Institute of Forest Science, Korea (Accession No. CDH052-054). The internal transcribed spacer (ITS) region of rDNA, beta-tubulin (TUB2) and a partial sequence of the actin (ACT) genes were amplified and sequenced for each of the isolates using the pair of primers, ITS1F/ITS4 (Gardes and Bruns 1993; White et al. 1990), T1/Bt2b (ODonnell and Cigelnik 1997; Glass and Donaldson 1995) and ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. A BLAST search in GenBank revealed that the sequences of ITS (OK631731-733), TUB2 (OK665927-929) and ACT (OK665930-932) showed sequence identities of 98.6 to 99.6% to Colletotrichum siamense sequences (FJ972613, FJ907423, FJ907438). A maximum likelihood tree, based on a combined dataset of ITS, ACT and TUB2 gene sequences for Colletotrichum spp., revealed that the three isolates were clustered with type specimens of C. siamense. To prepare larger quantities of inoculum for the pathogenicity, mycelial plugs bearing acervuli taken from 2% PDA were incubated in a conical flask containing 200 ml of 2% potato dextrose broth at 25°C on a rotary shaker at 150 rpm for two weeks. Spore concentration was adjusted to 1.0 × 104 ml-1 conidia of C. siamense (CDH054). A 10 to 15 ml of spore suspension was then sprayed on each leaf of 12 seedlings of 'Sinlyeong' walnut (three-year-old), while 7 seedlings were treated with sterile distilled water as a control. Each treated seedling was covered by a plastic bag to maintain moisture for one day. Inoculation trials were repeated twice, in August and September 2021. Symptoms identical to those observed in the field developed four to five days after the inoculations from which the inoculated pathogen was successfully re-isolated, fulfilling Koch's postulates. However, no symptoms were observed in the control. To our knowledge, this is the first report of anthracnose on J. regia caused by C. siamense in Korea. This indicates that disease occurrences must be further rigorously surveyed at the nation-wide scale to effectively control the disease in the country.

Cultural and morphological characteristics of Colletotrichum sublineolum isolates infecting sorghum in eastern Ethiopia

Colletotrichum sublineolum is the most destructive pathogen causing sorghum anthracnose worldwide. The fungus is known to have highly variable pathotypes. A characteristic study of pathogen is important to document the change occurring in population as variability in morphology indicates the existence of different pathotypes. Controlled condition experiment was conducted to examine cultural and morphological characteristics of C. sublineolum isolates infecting sorghum in eastern Ethiopia. Sorghum leaves showing symptoms of anthracnose were collected from five districts through survey. To study the characteristics of C. sublineolum, single-spore isolates representing isolate collection districts were selected from the stock cultures and cultivated on potato dextrose agar. Culture growth, colony color, elevations, texture and margin, conidial diameter and shape were used to characterize isolates. The isolates were varied significantly in many aspects. Colony colors were differed from light-gray to gray, purple-gray to cottony-gray, white to salmon-whit, plum-pink to beige and rosy brown on upper side of the petri dishes. The mean culture growth of C. sublineolum isolates showed highly significant (P < 0.01) variations among each other and ranged from 15 to 44 mm eight-days after incubation. Most of the isolates were produced hyaline, smooth walled, falcate conidia but without septa. Conidial diameter of C. sublineolum isolates showed variations with width and length ranged from 2.97 to 6.01 μm and 10.01–27.75 μm, respectively. Most isolates had smooth colony margin and few had undulated margin. This finding revealed that substantial variations were observed among C. sublineolum isolates and the existence of variable characteristic showed the presence of several sub-species of the pathogen infecting sorghum in different agro-ecologies of eastern Ethiopia.

Potensi Khamir Epifit Indigenus untuk Mengendalikan Colletotrichum capsici, Penyebab Penyakit Antraknosa pada Buah Cabai Merah

Anthracnose is an important disease in red chili caused by Colletotrichum capsici which can reduce productivity by up to 65%. Controlling using indigenous epiphytic yeasts has never been done. The study aimed to determine the ability of indigenous epiphytic yeast isolates to control C. capsici on red chili. The study was carried out at the Phytopathology Laboratory, Faculty of Agriculture, Universitas Andalas, from August 2019 - April 2020 in vitro and in vivo. The study used a completely randomized design (CRD) which consisted of 6 treatments with five different isolates (4 isolates from fruit and one from leaves) and a control. Parameters observed were macroscopic and microscopic characteristics of yeasts, C. capsici colony area, disease incubation period, and anthracnose symptoms. The results showed that the five epiphytic yeast isolates could suppress the growth of C. capsici, inhibit colony expansion between 27.09 – 59.11%, extend the incubation period for one day, and inhibit the expansion of anthracnose symptoms between 52.30 – 62.64%. Epiphytic yeast isolate KB1 derived from fruit has the highest inhibition.

First Report of Anthracnose Caused by Colletotrichum sansevieriae on Sansevieria trifasciata var. laurentii in China.

Sansevieria trifasciata var. laurentii (De Wild.) N.E. Brown, commonly known as variegated snake plant or variegated mother-in-law's tongue, is a popular landscape and house plant. In September and October 2019, the obvious leaf spot symptoms were observed on the plants in a 0.2 hm2 of nursery in Qingdao city of China with incidence of 55%. The disease usually starts from the tip or edge of the leaf, initially have slightly water-soaked semi-circular or round brown lesions, which gradually expanded and coalesced into irregular shapes about 3-8 cm in diameter. Grayish brown sunken spots with dark margins that evolve into concentric rings of acervuli which were characteristic of anthracnose, and orange sticky conidial masses were observed under the moist condition. The leaves with typical anthracnose symptoms were collected and deposited in the herbarium of Qingdao agricultural university under accessions no. QDHB074-QDHB087. Subsequently 20 isolates with the same colony and morphological characteristics were obtained from ten diseased leaves by placing surface-sterilized tissue pieces with typical spots on potato dextrose agar (PDA). Colonies are floccose with grayish-white to dark olivaceous gray color, and gray black on the reverse after 14 days at 28°C. Straight conidia [15.0 to 27.5 × 3.5 to 7.0 μm in size (average 18.2 × 6.1 μm) (n = 50)] were cylindrical, aseptate, hyaline, slippery surface, most with one tapering end and the other oval. Setae were black, 185-230 μm in length, with a thin tip and septate in the middle. Appressoria [6.5 to 7.3 × 7.8 to 9.2 μm in size (average 6.8 × 8.1 μm) (n = 15)] were black to dark brown, solitary, spherical with smooth wall. The fungal isolates were identified as Colletotrichum sansevieriae Nakamura (Nakamura et al. 2006), based on the morphological characteristics. To confirm the identification, the internal transcribed spacer (ITS) and calmodulin (CAL) regions of a representative isolate HWL-1016 were amplified by primers ITS1/ITS4 (White et al. 1990) and CMD5/CMD6 (Weir et al. 2012), respectively. The 549 bp ITS (MN922517) and 597 bp CAL (OM994078) sequences had respectively 100% and 99.30% identity with the sequences from holotype species of C. sansevieriae MAFF 239721 (no. NR_152313 and LC180125). Phylogenetic tree based on ITS and CAL sequences respectively or jointly constructed by PAUP4.0 (Swofford 2002) revealed that the fungus in this study clustered with C. sansevieriae isolates (NR_152313, KC790947, HQ433226, JF911349, MN386823). Pathogenicity test of isolate HWL1016 was evaluated on five 3- to 4-month-old potted S. trifasciata var. laurentii under greenhouse conditions (27±2 °C, 16-hr light/8-hr dark photoperiod, 80% relative humidity). Conidial suspension (1×106 conidia/mL) of the isolated fungus from PDA colonies cultured for 15 days and sterile distilled water (as control) were sprayed on pin-pricked surface-sterilized (70% alcohol) leaves of potted plants, respectively. Three replications (three plants) were done for each treatment, and the experiment was repeated twice. The inoculated plants were covered with plastic films for 2 days and obvious water-soaked wounds were observed on the sixth day. After 16 days, the symptoms of the inoculated plants were similar to those in the nursery, with disease incidence reached 100%, while controls remained symptomless. C. sansevieriae was subsequently reisolated from the symptomatic tissues. Anthracnose on S. trifasciata var. laurentii caused by C. sansevieriae has been reported in Australia, Iran, Japan, Malaysia (Kee et al. 2020), South Korea, USA (Talhinhas & Baroncelli 2021), India (Gautam et al. 2012) and Thailand (Li et al. 2020). To our knowledge, this is the first report of C. sansevieriae causing anthracnose on S. trifasciata var. laurentii in China. This study will contribute to guide effective management based on pathogen.

Morphological characterization and pathogenicity of Colletotrichum aenigma and C. siamense causing anthracnose on Euonymus japonicus in Beijing, China

AbstractEuonymus japonicus plays important roles in the process of urban landscape construction as an evergreen shrub. Anthracnose is an important limiting factor affecting the healthy growth of E. japonicus, seriously influencing improvement of urban landscape ecosystems. However, the Colletotrichum species associated with anthracnose on E. japonicus are unclear. In this study, diseased leaves of E. japonicus with typical anthracnose symptoms were collected in five nurseries in Beijing, China. Among 45 Colletotrichum isolates obtained, there were two distinct morphotypes, which were identified as C. aenigma or C. siamense by morphological characteristics and multilocus phylogenetic analysis. The growth rates of these two species were determined at different incubation temperatures, and the results showed that both species grew well at 10–35°C, particularly at 25 or 30°C, while C. aenigma isolates were adapted to high growth rates at a wider range of temperatures than C. siamense. Pathogenicity assays indicated that the two species showed varying degrees of pathogenicity on E. japonicus. Wounding was conducive to the pathogenicity of C. aenigma or C. siamense on E. japonicus either by inoculation with mycelial discs or conidial suspensions. This study presents the first report of C. aenigma causing anthracnose on E. japonicus worldwide and of C. siamense in north China causing anthracnose on E. japonicus. The morphological features of Colletotrichum spp. associated with anthracnose on E. japonicus were also compared in detail in this study. The findings provide a contribution to the prevention and control of anthracnose on E. japonicus by better understanding the species involved.

Colletotrichum truncatum-A New Etiological Anthracnose Agent of Sword Bean (Canavalia gladiata) in Southwestern China.

Anthracnose is a disease caused by Colletotrichum species. They are well known as major plant pathogens, and a black stem disease, specifically caused by Colletotrichum truncatum and primarily infecting sword bean (Canavalia gladiata), was observed in the Yunnan province, China. To aid disease management and to determine pathogenic characteristics, the species causing the leaf spot disease of hairy vetch was verified as C. truncatum. A sequence analysis of the ITS, ACT, GAPDH, and HIS3 genes was conducted, as well as morphological and cultural characteristics, to identify this Colletotrichum species, which has curved conidia. C. truncatum isolates from sword bean formed a distinctive group among Colletotrichum species, including those that infect other forage and field crops. Artificially inoculated sword bean seedlings showed typical symptoms of anthracnose, which were similar to field observations. To the best of our knowledge, this is the first report of C. truncatum causing black stem disease on sword beans in China.

Comparative transcriptomic provides novel insights into the soybean response to Colletotrichum truncatum infection.

Soybean (Glycine max) is among the most important crops in the world, and its production can be threatened by biotic diseases, such as anthracnose. Soybean anthracnose is a seed-borne disease mainly caused by the hemibiotrophic fungus Colletotrichum truncatum. Typical symptoms are pre- and post-emergence damping off and necrotic lesions on cotyledons, petioles, leaves, and pods. Anthracnose symptoms can appear early in the field, causing major losses to soybean production. In preliminary experiments, we observed that the same soybean cultivar can have a range of susceptibility towards different strains of C. truncatum, while the same C. truncatum strain can cause varying levels of disease severity in different soybean cultivars. To gain a better understanding of the molecular mechanisms regulating the early response of different soybean cultivars to different C. truncatum strains, we performed pathogenicity assays to select two soybean cultivars with significantly different susceptibility to two different C. truncatum strains and analyzed their transcriptome profiles at different time points of interaction (0, 12, 48, and 120h post-inoculation, hpi). The pathogenicity assays showed that the soybean cultivar Gm1 is more resistant to C. truncatum strain 1080, and it is highly susceptible to strain 1059, while cultivar Gm2 shows the opposite behavior. However, if only trivial anthracnose symptoms appeared in the more resistant phenotype (MRP; Gm1-1080; Gm2-1059) upon 120 hpi, in the more susceptible phenotype (MSP; Gm-1059; Gm2- 1080) plants show mild symptoms already at 72 hpi, after which the disease evolved rapidly to severe necrosis and plant death. Interestingly, several genes related to different cellular responses of the plant immune system (pathogen recognition, signaling events, transcriptional reprogramming, and defense-related genes) were commonly modulated at the same time points only in both MRP. The list of differentially expressed genes (DEGs) specific to the more resistant combinations and related to different cellular responses of the plant immune system may shed light on the important host defense pathways against soybean anthracnose.

Competition between Colletotrichum species reduces anthracnose symptom development in Dioscorea alata yams: Potential for biopriming and breeding

AbstractEcological interactions are a fairly overlooked component in agriculture, despite their known potential in productivity, pest and disease regulation. In this study, we assessed whether a potential low pathogenic congeneric competitor of Colletotrichum gloeosporioides could decrease disease onset in yams. We set up an experiment with artificial drop inoculation on detached leaves, with individual controls and treatments as either co‐inoculation or inoculation with a harmless competitor 24 h prior to inoculating the pathogen. Our results demonstrate that direct admixing translated into accrued competition and disease symptoms, while prior inoculation of the competitor tended to decrease symptom development. There was variation in how varieties responded to treatments and in strain competitive ability altering the protective effect, but nevertheless these results suggest that incorporating such interactions in breeding programmes or working towards increased use of commensals in agriculture might confer improved disease control for crops.

Diversity and Resistance to Thiophanate-Methyl of Colletotrichum spp. in Strawberry Nursery and the Development of Rapid Detection Using LAMP Method

Anthracnose is a devastating fungal disease in strawberry nurseries. Multiple Colletotrichum species are responsible for strawberry anthracnose. In this study, 105 Colletotrichum isolates were obtained from strawberry seedlings with anthracnose symptoms in fifteen nurseries located in Zhejiang province, China, and were classified based on multilocus sequence and morphological characteristic analyses. Analysis of ITS, ACT, CAL, CHS-1, and GAPDH revealed that four species within C. gloeosporioides species complex, including C. siamense (56 isolates, 53.3%), C. fructicola (37 isolates, 35.2%), C. gloeosporioides (7 isolates, 6.7%), and C. aenigma (5 isolates, 4.8%), were detected in diseased seedlings. Thiophanate-methyl is one of the benzimidazole fungicides, and has long been used to control strawberry anthracnose in China. Here, thiophanate-methyl resistance of Colletotrichum isolates was determined by the minimum inhibitory concentration (MIC) method. Our results indicated that the resistance frequency was up to 96.2%, containing 94.3% of highly resistant isolates. Only four sensitive isolates (two C. fructicola, one C. gloeosporioides, and one C. siamense isolates) and two moderately resistant isolates (one C. aenigma isolate and one C. siamense isolate) were detected. Our data indicated that the high resistance was mainly caused by the E198A mutation in the β-tubulin protein. In addition, F200Y (TTC→TAC) in the β-tubulin protein were detected in two moderately resistant isolates. Based on the point mutation at codon 198 (GAG→GCG) in the β-tubulin gene of Colletotrichum isolates, we developed a loop-mediated isothermal amplification (LAMP) assay to rapidly detect the E198A mutants. Collectively, our study indicated that four species within the C. gloeosporioides species complex were associated with anthracnose symptoms in strawberry nurseries in Zhejiang province, and serious resistance was widespread in each Colletotrichum species.

First Report of Colletotrichum chrysophilum Causing Papaya Anthracnose in Mexico.

Anthracnose, caused by Colletotrichum spp., is the most important fungal disease of papaya (Carica papaya L.) worldwide. In March 2020, mature papaya fruit (cv. Maradol) showing typical symptoms of anthracnose were observed in an orchard located in Pinotepa Nacional, Oaxaca, Mexico. Disease incidence of 100 papaya plants surveyed in the orchard was estimated at about 45%. Initially, small and water-soaked lesions appeared on the fruit surface, which later enlarged to circular sunken lesions with translucent light brown margins. On advanced infections, salmon-pink masses of spores were observed on the lesions. Twenty Colletotrichum-like colonies were consistently isolated on potato dextrose agar (PDA) medium at 25°C in the dark for 6 days and 10 monoconidial isolates were obtained. An isolate was selected as representative for further characterization. The isolate was deposited as CPM-H4 in the Culture Collection of Phytopathogenic Fungi of Plant Pathology Laboratory of the CIIDIR-Oaxaca of the Instituto Politécnico Nacional. On PDA, the colonies were initially light grey then later became dark grey with orange conidial masses after incubation for 7 days. Conidia (n= 50) were hyaline, aseptate, cylindrical with rounded ends, and measured 10.2 to 13.6 × 4.1 to 5.3 μm. Appressoria (n= 20) were mostly simple, solitary and smooth-walled, dark brown, and clavate, measuring 6.8 to 14.8 × 5.5 to 7.7 μm. Based on morphology, the isolate was tentatively identified as belonging to 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), β-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and chitin synthase (CHS-1) genes were amplified (Weir et al. 2012), and sequenced. The sequences were deposited in GenBank (accessions nos. OM965612 (ITS), OM959540 (ACT), ON065005 (TUB2), ON065003 (CHS-1), ON065004 (GAPDH). A phylogenetic tree based on Bayesian inference and including published ITS, ACT, TUB2, GAPDH, and CHS-1 sequence dataset for Colletotrichum spp. was constructed. The multilocus phylogenetic analysis clearly distinguished the isolate CPM-H4 as Colletotrichum chrysophilum. Pathogenicity of the fungus was verified on 10 healthy papaya fruits (cv. Maradol) without wounds. A drop of a conidial suspension (1 × 105 spores/ml) was placed on three locations on each fruit. Ten control fruit were treated in the same way but with sterilized water. The fruits were kept in a moist plastic chamber at 25°C and 12 h light/dark for 8 days. The pathogenicity test was repeated twice. All inoculated papaya fruits developed sunken necrotic lesions 6 days after inoculation, whereas no symptoms were observed on the control fruits. The fungus was consistently re-isolated only from the diseased fruits and found to be morphologically identical to the isolate used for inoculation, fulfilling Koch´s postulates. Colletotrichum chrysophilum has been previously reported to cause anthracnose on mango (Fuentes-Aragón et al. 2020a), avocado (Fuentes-Aragón et al. 2020b), and banana (Fuentes-Aragón et al. 2021) in Mexico; however, to our knowledge, this is the first report of C. chrysophilum causing papaya anthracnose in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species associated with papaya in Mexico through subsequent phylogenetic studies as well as to monitor the possible movement and distribution of this pathogen into other Mexican regions.

Potensi Ekstrak Daun Sirih dan Rimpang Lengkuas dalam Menekan Pertumbuhan Colletorichum gloeosporiodes Skala In Vitro

Colletotrichum gloeosporioides is the cause of anthracnose disease in chili both pre-harvest and post-harvest. Various control efforts continue to be developed to overcome these problems. The purpose of this study was to test the potency of betel leaf extract and galangal rhizome to suppress the growth of C. gloeosporioides on an in vitro scale. This study used a completely randomized design (CRD), consisting of 3 treatments, namely betel leaf extract, galangal rhizome extract, and control. Extracts of betel leaf and galangal rhizome were each prepared at a concentration of 25%, while pure cultures of C. gloeosporioides were isolated from chili peppers with anthracnose symptoms. Then 3 mL of each extract was mixed in 7 mL of Potato Dextrose Agar (PDA) media until homogeneous. Furthermore, the culture of C. gloeosporioides was inoculated on PDA media that had been treated with extract and without extract as a control. The growth diameter of C. gloeosporioides was then observed at 7 days after inoculation. The results showed that the treatment of betel leaf extract and galangal rhizome significantly affected the growth suppression of C. gloeosporioides compared to controls. Galangal rhizome extract was the best treatment in suppressing the growth of C. gloeosporioides with an efficacy of 100%, while betel leaf extract still had a low efficacy of 35%.

First Record of Colletotrichum anthrisci Causing Anthracnose on Avocado Fruits in Chile.

Anthracnose caused by Colletotrichum species is one of the most frequent and damaging fungal diseases affecting avocado fruits (Persea americana Mill.) worldwide. In Chile, the disease incidence has increased over the last decades due to the establishment of commercial groves in more humid areas. Since 2018, unusual symptoms of anthracnose have been observed on Hass avocado fruits, with lesions developing a white to gray sporulation. Morphological features and multi-locus phylogenetic analyses using six DNA barcodes (act, chs-1, gapdh, his3, ITS, and tub2) allowed the identification of the causal agent as Colletotrichum anthrisci, a member of the dematium species complex. Pathogenicity was confirmed by inoculating healthy Hass avocado fruits with representative isolates, reproducing the same symptoms initially observed, and successfully reisolating the same isolates from the margin of the necrotic pulp. Previously, several Colletotrichum species belonging to other species complexes have been associated with avocado anthracnose in other countries. To our knowledge, this is the first record of C. anthrisci and of a species of the dematium species complex causing anthracnose on avocado fruits in Chile and worldwide.

First report of Colletotrichum fioriniae causing anthracnose on the fruit of omija (schisandra) in South Korea.

Omija or Schisandra (Schisandra chinensis Baillon) is one of Korea's most unique fruits and have been used in herbal medicine. It is also used for making cold drinks during summer and hot tea during winter. Among the different diseases, anthracnose poses great threat for omija production as it reduces the yield and market quality of fruits. In September 2021, severe anthracnose symptoms were observed in some commercial farmer fields (36°36'14.9"N 127°59'31.3"E) in Mungyeong and disease incidence was approximately 30 to 35%. The symptoms on fruits were manifested as small sunken lesions. The presumed fungi were isolated from necrotic tissues on potato dextrose agar (PDA) and purified using the single spore isolation technique (Cai et al. 2009; Hassan et al.2019). A total of 20 morphological similar isolates were obtained from 18 sampled fruits. The cultures were incubated at 25°C in the dark for 5 to 7 days. The colonies were pink with white aerial mycelium on the upper side and pink with black zones on the reverse. All the colonies produced fusiform conidia measuring 12.7 to 17.5 × 4.7 to 7.4 μm (n=50). The appressoria were brown, globose to ellipsoid and measuring 6.3 to 12 × 5.9 to 9.7 μm (n=30). Morphological characteristics of present isolates were in concordance with that of Colletotrichum acutatum sensu lato (Damm et al., 2012). For molecular identification, genomic DNA of five representative isolates was extracted for sequencing of target markers and the phylogenetic tree was constructed. For amplification and sequencing of internal transcribed spacers (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT) chitin synthase-1 (CHS-1), histone 3 (HIS3) and beta-tubulin (TUB2), the primer pairs ITS1/ITS4, GDF1/GDR1, ACT-512F/ACT-783R, CHS1-79F/CHS-1-354R, CYLH3F /CYLH3R and BT2a /BT2b were used respectively (Damm et al., 2012). The resulting sequences were deposited in GenBank with accession numbers LC680890 to LC680914 and LC687627 to LC687631. The maximum likelihood tree based on concatenated sequences of the five genes shows that present isolates were grouped in the C. fioriniae. Pathogenicity on red schisandra fruits was confirmed using the two isolates OR1 and O59. Fruit clusters (20-30 fruits/ cluster) were surface sterilized by dipping them in 1% NaOCl for 3 minutes, rinsing in sterile distilled water, and placing them in a sterile plastic box containing moist paper tissues. All the fruits in each cluster were inoculated with 10 μl of 106 conidia/ml suspensions of each isolate by following either the wounded or un- wounded method (Hassan et al.2019). Fruit clusters treated with sterile distilled water served as a control. Three fruit clusters were inoculated per isolate for each method. With the wounding method, all the inoculated fruits showed typical anthracnose symptoms after 4 days of incubation at 25 ± 1°C in the dark, while the control and un- wounded fruits remained symptomless. This test was repeated twice. The species C. fioriniae were reisolated from the infected fruit. The morphology and ITS sequences of the reisolated fungi were consistent with the original one, fulfilling Koch's postulates. The species C. fioriniae have been described as a causal agent of anthracnose of numerous hosts worldwide including South Korea (Damm et al., 2012; Hassan et al.2019; Oo et al. 2016; Lee et al. 2020). To our knowledge, this is the first report of C. fioriniae as causal agent of omija anthracnose in South Korea. As this disease results in deterioration of fruits quality, management practices should be tailored to address C. fioriniae management. Acknowledgments This research was supported by Kyungpook National University Research Fund, 2021. References Cai, L., et al. 2009. Fungal Divers. 39:183. Damm, U., et al. 2012. Stud. Mycol. 73:37. Hassan, O., et al. 2019. Sci. Rep. 9:12089. Lee, D., et al. 2020. Mycobiology. 48: 210. Oo, M.M., et al. 2016. Mycobiology. 44: 325.

First Report of Colletotrichum nymphaeae CausingWalnut Anthracnose in China.

Walnut(Juglans regiaL.) is a high quality woody nut and edible oil tree with a planting area of about 5,000,000 hectare in China. Walnut anthracnose is a serious disease, infecting approximately 50% of the fruits and causing a great yield losses (Wang et al. 2016). In 2019 to 2020, walnutfruits with anthracnose symptoms were collected from walnut orchards in province of Hubei, Sichuan procinve and Chongqing municipality, China. Symptoms on fruits were circular or subcircularor irregular shaped, with brown to black water soaked and sunken lesions. The black lesions enlarged and amalgamated into large necrotic areas. The older spots in the center became blackish with acervuli causing the complete mummification of the fruit, and orange conidial masses appeared under wet conditions. Necrotic tissues of the fruits were sterilized in 75% ethanol solution for 30 s, then sterilized in 4% sodium hypochlorite for 1min, and washed 3 times with sterile distilled water. The tissues were put on potato dextrose agar (PDA) and incubated at 25℃. Pure cultures were obtained by single-spore culturing method and the isolate HBBK4-4 was deposited into the China's Forestry Culture Collection Center (CFCC 57388). On PDA, the colonies were cottony, white to pale gray with aerial mycelium on the upper side and pink with black spots on the reverse. The mycelial growth rate was 9.6 mm/day at 25°C. Conidia were 1-celled, colorless, smooth-walled, straight, cylindrical to cylindrical-clavate with acute ends, 12.5 to 18.2 × 3.9 to 5.4 μm (mean 15.3 ± 3.7 × 4.9 ± 0.6 μm,n= 40). Most conidia germinated and developed one pleurogenous, 1-celled appressorium. Appressoria were single, medium brown, smooth-walled, ovate to ellipsoid, 5.4 to 7.8 × 5.4 to 7.8 μm (mean 6.7 ± 0.6 × 6.3 ± 0.5 μm,n= 30). These morphological characteristics were in concordance with published descriptions of Collectotrichum acutatum species complex. To further confirm the identity, internal transcribed spacer (ITS), beta-tubulin (TUB2), chitin synthase 1 (CHS-I) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified and sequenced (Damm et al. 2012). The ITS (OM189549) and TUB2 (OM273642) sequences of isolate HBBK4-4 showed 100% similarity, and GAPDH (OM249791) and CHS-1 (OM273641) sequences showed 98.7% and 99.6% similarity withC. nymphaeaeCBS100064 respectively. A maximum likelihood phylogenetic tree was generated based on combining all sequenced loci in MEGA5. 18 isolates including HBBK4-4 fell in theC. nymphaeaeclade with 96% bootstrap support. To verify Koch's postulates, six isolates were used for pathogenicity test, and 20 healthy fruits and 15 fully expanded leaves for each isolate were inoculated with 5-mm-diameter mycelial plugs. Controls consisted of detached premature fruits inoculated with a PDA plug without the fungus. Six days after inoculation, all fruits and leaves developed anthracnose symptoms similar to those observed in the field, while the controls remained healthy. The pathogenicity tests were repeated twice with the same results. The morphology of the reisolated fungi was consistent with the inoculated one, fulfilling Koch's postulates.The isolate HBBK4-4 was identified as C. nymphaeae, based on the description byDamm et al. (2012). The species C. nymphaeae has been previously reported to cause severe anthracnose on walnut in France (Da Lio et al., 2018), Brazil (Savian et al., 2019) and Italy (Luongo et al., 2022). To our knowledge, this is the first report ofC. nymphaeaeas a pathogen ofwalnutanthracnose in China. The result provided crucial information for epidemiologic studies and management of this disease.

REAÇÃO DE ACESSOS DE FEIJÃO-FAVA (Phaseolus lunatus L.) A Colletotrichum truncatum

ABSTRACT Lima bean (Phaseolus lunatus) can be considered as an alternative income and food supply to farmers of the Brazilian Northeast. This crop has faced serious sanitary problems. Among the most important diseases that attack this crop, the anthracnose caused by Colletotrichum truncatum requires greater attention. This study aimed to determine the resistance of lima bean accessions to the isolates of C. truncatum. Detached leaves were used from ten lima bean accessions, originated from production fields in Paraíba State - Brazil. They were artificially inoculated with ten isolates of C. truncatum. The isolates were grown on bean-dextroseagar medium under temperature 27 ± 2 °C and a photoperiod of 12 hours, for fourteen days. The evaluations of the accessions’ resistance to the isolates were performed at 5, 7, 9, 11 and 13 days after inoculation (DAI), adopting a scale of notes, and the results were transformed in the area under the disease progress curve. The research was carried out in completely randomized design, in a 10x10 factorial arrangement (accessions x isolates) with 12 replications. The first symptoms of anthracnose on the detached leaves appeared from the third day after inoculation. The accessions were grouped from highly to moderately resistant or susceptive. The aggressiveness among C. truncatum isolates varied depending on the genetic variability of the lima bean accessions used. Accessions with significant resistance levels to anthracnose can be used as resistance sources in future breeding programs.