Colletotrichum Fructicola Research Articles

Colletotrichum fructicola Causal agent of Shot-Hole Symptoms on Leavesof Prunus sibirica in China.

Prunus sibiricaL. (Siberian apricot) is a member of the Rosaceae family and an ecologically important tree species in China (Buer et al., 2022). Shot hole symptoms on the leaves were observed in five Siberian apricot groves in Chengdu (103.81 E, 30.97 N), Sichuan province in July 2020. The symptoms first appeared as small purplish-brown spots with yellow rings around them. As the disease progressed, the damaged area (diameter 1.5-3.0 cm) became necrotic and fell off. The disease incidence was about 60% and the disease index was 28.6 of leaves in the grove. in most severe cases. Fifteen symptomatic leaves were collected from 5 different trees in an orchard. Pathogen isolation was performed from symptomatic leaf tissue (5 × 5 mm) though surface disinfection (in 70% ethanol and 2% NaClO) and incubation on Potato Dextrose Agar (PDA) at 28℃ for 3 days. Overall 10 isolates with similar colony morphology wereobtained from the 15 infected tissue pieces, and three representative isolates (XCK 2-4) were selected for further study. Colonies of the isolates on PDA were initially cottony, pale white to grayish-green with abundant aerial hyphae and produced conidial masses after 7 days. Conidiogenous cells were clavate and aggregated in acervuli. Conidia were smooth-walled, single-celled, straight, and slightly obtusely rounded at both ends, 12.8 to 18.7 × 4.3 to 5.7 μm in size (Fig. 1). The morphological characteristics of the three isolates were consistent with the description of species in the Colletotrichum gloeosporioides complex. DNA was amplified using the following primers pairs for the internal transcribed spacer (ITS) region of rDNA and partial sequences of beta-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and translation elongation factor (TEF-1), respectively: ITS1/ITS4, T1/Bt2b, GDF/GDR, CHS-F/CHS-R, and EF-F/EF-R (Vieira et al., 2014). Accession numbers (MW228049, MW284974, MW284976, MW284975 and MW284977, respectively) were obtained afterepositing all the resulting sequences in GenBank. Nucleotide blast showed 99 to 100% identities with Colletotrichum fructicola (GenBank accessions nos. MZ961683, MW284974, MN525881, MN525860, MF627961). Phylogenetic analysis of combined ITS-TUB-GAPDH genes using the Mrbayes inference method showed that the three isolates clustered with three reference isolates of C. fructicola as a distinct clade(Fig. 2). To verify Koch's postulates, ten 3-year-old healthy potted plants ofP.sibirica were inoculated by spraying a conidial suspension (6 × 105 conidia/mL) of isolate XCK2 on both sides of leaves, and the control leaves were sprayed with sterile water. Then, all treatments were placed in a moist environment (25±2°C, 80% relative humidity, natural light). The inoculated plants showed typical symptoms of plants with natural infections, while the controls remained asymptomatic after 14 days. The pathogen C. fructicola was re-isolated from all inoculated plants, and the culture and fungus characteristics were the same as those of the original isolate. Colletotrichum fructicola was not isolated from the control plants. The results indicated that C. fructicola is the causal agent of the disease. Colletotrichum fructicola was reported as a leaf pathogen on Camellia chrysanthain China (Zhao et al., 2021). This is the first report of C. fructicola causing P. sibirica leaf shot-hole in the world. The identification of C. fructicola could provide relevant information for applying management strategies and research on the Siberian apricot disease.

First report of anthracnose on spotted laurel caused by Colletotrichum fructicola in South Korea.

Spotted laurel (Aucuba japonica) is a popular ornamental bush (it has two-colored leaves and red berries) and is used outdoors and indoors for decoration in South Korea. Anthracnose reduces the aesthetic value of spotted laurel leaves. In August 2022, anthracnose symptoms were observed on leaves in a park at Jeju Island, South Korea. Approximately 55% of bushes were infected by this disease. Symptoms consisted of round or irregular lesions that initially appeared as black spots and coalesced into larger, black lesions covering whole leaves and twigs. Entire leaves wither and finally die. To identify the putative causal agent, 12 affected leaves were collected, placed in a plastic box containing moist tissue, and incubated at 25 ºC in the dark to obtain conidial mass. Conidial masses were produced on leaf lesions after 2 days, and then 12 morphologically similar fungal isolates were recovered following single the spore isolation technique on solid potato dextrose agar (PDA) (Cai et al. 2009). Ten-day-old colonies were olivaceous gray with immersed perithecia on the upper side and black at the center on the reverse side. Conidia were aseptate, cylindrical with round ends and measured 14.9 - 22.7 × 5.5 - 9.4 μm (n = 80). Appressoria were brown, irregular in shape, and 7.0 - 16.1 × 5.00 - 9.9 μm (n = 50). Asci were eight-spored, banana-shaped, and measuring 60.8 - 123.1 × 13.00 - 18.9 μm (n = 30). Hyaline ascospores were single-celled, curved or straight with round ends, and ranged in size was 15.5 - 23.3 × 5.1 - 11.8 μm (n = 50). The morphological characteristics of the isolates overlapped with those of Colletotrichum species within the C. gloeosporioides complex, including Colletotrichum fructicola (Weir et al. 2012). Five genomic DNA loci of the isolates, including the partial ITS rDNA region, ACT, GAPDH, TUB, and ApMat genes, were amplified and sequenced using ITSF1/ITS4, ACT-512F/ACT-783R, GDF/GDR, T1/Bt2b, and AM-F/AM-R, respectively (Silva et al. 2012; Weir et al. 2012). The resulting consensus sequences were deposited in the GenBank and the accession numbers (ITS = LC739331- LC739334, TUB = LC739335- LC739338, GAPDH = LC739339- LC739342, ACT = LC739343 -LC739346, ApMat = LC742925 - LC742928) were obtained. A maximum phylogenetic tree was constructed based on the combined data sets of ITS, ACT, GAPDH, TUB, ApMat sequences. The isolates were clustered with reference isolates of C. fructicola (isolates ICMP18581). The pathogenicity test was performed on uninfected, healthy spotted laurel cuttings in the pot. Five leaves per seedling were selected, surface sterilized with 70% ethanol, and rinsed with sterile distilled water (SDW). A sterile pin was used to make 3 to 4 wounds on each side of the leaf from the midrib. 10 μl of spore suspension per wound spot (1 × 106 spores/ml) was applied on the wounds of one site from midrib, and SDW was placed on the wounds of other site as a control. The treated seedlings were covered with sterile plastic bag and kept in a 12-h fluorescent light/dark cycle under greenhouse conditions at 25 ± 2°C and 80% relative humidity. Two seedlings were inoculated with a single isolate, and this experiment was repeated twice. Circular or irregular lesions appeared after 5 days of inoculation, while the control remained asymptotic. Koch's postulates were fulfilled by reisolating and reidentifying the causal agent from the lesions of inoculated leaves. Colletotrichum fructicola has been reported as the causal agent of anthracnose on mango (Joa et al. 2016), apple (Kim et al. 2018), grapes (Lim et al. 2019), peaches (Lee et al. 2020), and hybrid pear (Choi et al. 2021) in South Korea. To the best of our knowledge, it is the first report of C. fructicola causing anthracnose on spotted laurel. This study will be helpful to develop effective management strategies to minimize leaf lesions.

First report of Neopestalotiopsis rosae causing leaf blight on Shatangju in southern China.

Shatangju (CitrusreticulataBlancocv.Shatangju) belongs to genus Citrus and was cultivated extensively in southern China. In April 2022, a leaf blight-like symptom (firstly brown spots appeared on infecting leaves, then these brown spots extended, finally the whole leaves displayed blight-like symptom) was observed on 5%~10% of Shatangju seedlings (around five hundreds in total) in an orchard located in Wuhan city, Hubei, China. Diseased leaves from three seedlings were collected and cut into pieces (0.2 to 0.5 cm). These pieces were surface-sterilized using 75% ethanol for 3 min, rinsed with sterile distilled water for several times, then placed on potato dextrose agar (PDA) and incubated at 26°C with 12-h light/dark cycle. Over 20 pieces plated, wherein 30% were identified as Colletotrichum fructicola, 60% as Neopestalotiopsis spp., and 10% developed saprophytes. C. fructicola was a known pathogen on citrus, thus Neopestalotiopsis spp. was further investigated. Eight single-conidium colonies of the Neopestalotiopsis spp. were obtained, wherein STJ-8 was chosen as a representative for further study. The average growth rate of STJ-8 was 15.1±0.5 mm/day (n=5). Fungal colonies produced white cottony mycelium with abundant black acervuli distributed in concentric rings 6-8 days after planting, which ranged from 342.3 to 710.5 μm in diameter (n=100). Conidia were fusoid, five cells, four septa with average dimensions of 25.36×5.47 μm (n=100). Basal and apical cells were hyaline, wherein three middle cells were brown with darker septa. The apical cell was cylindrical with two to three transparent accessory filaments (13.7 to 30.5 µm in length, n=80). Basal cell was conic with an appendage (4.1 to 8.8 µm in length, n=40). Partial sequences of internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α), and β-tubulin (TUB2) were amplified with reported primers (White et al. 1990; Lee et al. 2006; Maharachchikumbura et al. 2014), sequenced, and submitted to GenBank (accession nos. ITS: OP236541; TEF-1α: OP250124; TUB2:OP263094). BLASTn results showed 100% identity with the corresponding sequences of Neopestalotiopsis rosae. A multilocus phylogenetic analysis showed STJ-8 was closest to N. rosae. Thus, STJ-8 was identified as N. rosae. Pathogenicity tests were performed on one-year-old Shatangju seedlings and detached primary leaves by inoculating needle-wounded leaves with seven days old 5-mm mycelial plugs/acervuli (about 5000 spores) of STJ-8. Control seedlings/leaves were inoculated with 5-mm PDA plugs/sterile water drops. All inoculated detached leaves were cultured at same the place with STJ-8 cultured, while inoculated seedlings were put in a growth chamber at 26°C under a 16-h light/dark cycle (60% humidity). Symptoms developed on all inoculated leaves (except healthy control) 2 and 4 days post-inoculation by mycelial plugs and acervuli, respectively. N. rosae was re-isolated from the inoculated leaves, confirming Koch's postulates. N. rosae has been reported to cause diseases on various plants worldwide (Rebollar-Alviter et al. 2020; Xavier et al. 2021; Lawrence et al. 2022). In China, N. rosae has been reported to cause leaf spot/blight on pecan and strawberry (Wu et al. 2021; Gao et al. 2022), which caused great loss on these crops. To our knowledge, this is the first report of N. rosae causing leaf disease on citrus. Our study is important for developing control strategies against N. rosae in future.

First Report of Anthracnose on Tetrapanax papyriferus Caused by Colletotrichum fructicola in China.

Tetrapanax papyriferus is an evergreen shrub native to China and traditionally used as a herbal medicine (Li et al., 2021). In September 2021, a serious leaf spot disease with symptoms similar to anthracnose was extensively observed on T. papyriferus in Shibing county (E 127°12'0", N 25°11'60"), Qiandongnan Miao and Dong Autonomous Prefecture, Guizhou province, China. Field surveys were conducted in about 1000 T. papyriferus plants in Shibing in September 2021. The incidence of the leaf spot on leaves was 45% to 60%, significantly reducing the quality of medicinal materials. The symptoms began as small yellow spots, developing a brown center and dark brown to black margin, and eventually the diseased leaves were wiltered and rotted. Symptomatic leaves were collected from 20 trees. Symptomatic tissue from diseased leaves was surface desinfected (0.5 min in 75% ethanol and 1 min in 3% NaOCl, washed three times with sterilized distilled water), small pieces of symptomatic leaf tissue (0.2 × 0.2 cm) were plated on potato dextrose agar (PDA) and incubated at 25°C for about 7 days (Fang. 2007). Three single-spore isolates were obtained (GUTC37, GUTC310 and GUTC311) and deposited in the collection of the Plant Pathology Deparment, College of Agriculture, Guizhou University, China (GUCC) (with the accession numbers, GUCC220241, GUCC220242, GUCC220243 respectively). These isolates were identical in morphology and in the sequences of internal transcribed spacer region [ITS], glyceraldehy-3-phosphate dehydrogenase [GAPDH], chitin synthase [CHS-1], actin [ACT], and calmodulin [CAL] genes (White et al. 1990; Carbone and Kohn 1999; Templeton et al. 1992). Therefore, the representative isolate GUTC37 was used for further analysis. The pathogenicity of GUTC37 was tested through a pot assay. Plants were inoculated by spraying a spore suspension (106 spores·ml-1) of isolated strains onto leaves until runoff, and the control leaves sprayed with sterile water. The inoculated plants were incubated in a growth chamber at 28 ℃ and 95% relative humidity for 10 days. Pathogenicity tests were repeated three times (Fang. 2007). The symptoms developed on the inoculated leaves, while control remained asymptomatic. The lesions were first visible 72 h after inoculation, and typical lesions like those observed on field plants appeared after 10 days. The same fungus was reisolated and identified based on the morphological characterization and molecular analyses from the infected leaves but not from the non-inoculated leaves. Results of pathogenicity experiments of isolated fungi fulfilled Koch's postulates. Fungal colonies on PDA were villiform, creamy-white or greyish, aerial mycelium pale grey, dense, surface partly covered with orange conidial masses. The conidia were abundant, oval-ellipsoid, aseptate, and 13.89 (11.62 to 15.21) × 5.21 (4.39 to 5.65) µm (n=50). Appressorium were greyish green, nearly ovoid to cylindrical, 9.64 (6.62 to 14.61) × 6.33 (5.45-7.72) µm (n=50). The morphological features were consistent with the descriptions of Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde (Prihastuti et al. 2009). The pathogen was identified to be C. fructicola by amplification and sequencing of the five genes. The sequences of the PCR products were deposited in GenBank with accession numbers OP143657 (ITS), OP177868 (GAPDH), OP177865 (CHS-1), OP278677 (ACT) and OP177862 (CAL). BLAST searches of the obtained sequences revealed 100% (509/509 nucleotides), 99.63% (269/270 nucleotides), 99.31% (287/289 nucleotides), 99.29% (280/282 nucleotides), and 99.86% (728/729 nucleotides) homology with those of C. fructicola in GenBank (JX010165, JX010033, JX009866, FJ907426, and JX009676, respectively). Phylogenetic analysis (MEGA 7.0) using the maximum likelihood method placed the isolate GUTC37 in a well-supported cluster with C. fructicola. To our knowledge, this is the first report of anthracnose on T. papyriferus caused by C. fructicola in Guizhou, China. This study provides valuable information for the identification and control of the anthracnose on T. papyriferus.

The m6 A reader MhYTP2 negatively modulates apple Glomerella leaf spot resistance by binding to and degrading MdRGA2LmRNA.

Glomerella leaf spot (GLS), caused by the fungal pathogen Colletotrichum fructicola, significantly threatens apple production. Some resistances to plant disease are mediated by the accumulation of nucleotide-binding site and leucine-rich repeat (NBS-LRR) proteins that are encoded by a major class of plant disease resistance genes (R genes). However, the R genes that confer resistance to GLS in apple remain largely unclear. Malus hupehensis YT521-B homology domain-containing protein 2 (MhYTP2) was identified as an N6 -methyladenosine RNA methylation (m6 A) modified RNA reader in our previous study. However, whether MhYTP2 binds to mRNAs without m6 A RNA modifications remains unknown. In this study, we discovered that MhYTP2 exerts both m6 A-dependent and -independent functions by analysing previously obtained RNA immunoprecipitation sequencing results. The overexpression of MhYTP2 significantly reduced the resistance of apple to GLS and down-regulated the transcript levels of some R genes whose transcripts do not contain m6 A modifications. Further analysis indicated that MhYTP2 binds to and reduces the stability of MdRGA2L mRNA. MdRGA2L positively regulates resistance to GLS by activating salicylic acid signalling. Our findings revealed that MhYTP2 plays an essential role in the regulation of resistance to GLS and identified a promising R gene, MdRGA2L, for use in developing apple cultivars with GLS resistance.

First Report of Anthracnose Leaf Spot Caused by Colletotrichum fructicola on Manglietia decidua (Magnoliaceae) in China

First Report of Anthracnose Leaf Spot Caused by <i>Colletotrichum fructicola</i> on <i>Manglietia decidua</i> (Magnoliaceae) in China

Cell Wall Integrity Mediated by CfCHS1 Is Important for Growth, Stress Responses and Pathogenicity in Colletotrichum fructicola

Camellia oleifera, a woody plant that produces edible oil, is indigenous to China. The devastating disease of anthracnose inflicts significant financial losses on Ca. oleifera. The primary causative agent of anthracnose on Ca. oleifera is Colletotrichum fructicola. Chitin, a pivotal constituent of fungal cell walls, assumes a critical function in their proliferation and maturation. To study the biological functions of chitin synthase 1(Chs1) in C. fructicola, the CfCHS1 gene knockout mutants, ∆Cfchs1-1 and ∆Cfchs1-2, and their complementary strain, ∆Cfchs1/CfCHS1, of C. fructicola were generated. Our results showed that the colony diameters of wild-type and complement-strain ∆Cfchs1/CfCHS1, mutant ∆Cfchs1-1 and ∆Cfchs1-2 cultured on the CM and MM medium were 5.2, 5.0, 2.2 and 2.4 cm and 4.0, 4.0, 2.1 and 2.6 cm, respectively, which were significantly smaller for the mutant than for the wild type and complement strain; the inhibition rates on the CM medium supplemented with H2O2, DTT, SDS and CR were 87.0% and 88.5%, 29.6% and 27.1%, 88.0% and 89.4%, and 41.7% and 28.7%, respectively, for the mutant strains, ∆Cfchs1-1 and ∆Cfchs1-2, which were significantly higher than those for the other two strains; the rate of hyphal tips with CFW fluorescence in ∆Cfchs1-1 and ∆Cfchs1-2 was 13.3% and 15.0%, which was significantly lower than those for the other two strains; the mutant strains, ∆Cfchs1-1 and ∆Cfchs1-2, lost the ability to produce conidia; the mutant strains showed weaker pathogenicity on wounded and unwounded Ca. oleifera leaves than the wild type and complement strain. The findings of this study suggest that CfChs1 plays a crucial role in the growth and development, stress responses, and pathogenicity of C. fructicola. Thus, this gene could be a potential target for developing novel fungicide.

Identification and Characterization of Colletotrichum Species Causing Tea-Oil Camellia (Camellia oleifera C.Abel) Anthracnose in Hainan, China

Camellia oleifera C.Abel, commonly known as tea-oil camellia, is a type of significant woody oil crop that is widely cultivated in southern China. During 2017–2021, a fungal foliar disease was detected in routine surveys in Hainan. However, diseases of tea-oil camellia are seldom reported in Hainan. In this study, 51 Colletotrichum spp. isolates were obtained from the symptomatic samples of tea-oil camellia, collected from three production sites located in Hainan. A polyphasic approach was applied to distinguish Colletotrichum species. All 51 isolates were primarily characterized morphologically, and six gene regions, including an internally transcribed space of ribosomal DNA (ITS), chitinsynthase (CHS-1), β-tubulin (TUB), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and manganese-superoxide (SOD2), were sequenced for each isolate. By combining morphological characterization with multilocus sequence analysis (MLSA) based on the six genes, the fungal isolates were identified, representing three Colletrotrichum species: C. fructicola, C. siamense, and C. cordylinicola. The most predominant species was C. fructicola. In pathogenicity tests on the tea-oil camellia cultivar (Reyan1), all collected isolates were pathogenic on tea-oil camellia leaves and were reisolated from symptomatic leaves. Colletotrichum fructicola was the most aggressive species on the attached leaves. This is the first report of C. cordylinicola affecting tea-oil camellia anthracnose worldwide. These results will improve our understanding of the pathogens and provide important insights on the diagnosis and efficient disease management of tea-oil camellia anthracnose.

First report of Colletotrichum fructicola causing anthracnose of Japanese brome (Bromus japonicus Thunb.) in China

First report of Colletotrichum fructicola causing anthracnose of Japanese brome (Bromus japonicus Thunb.) in China

The CfAtg5 Regulates the Autophagy and Pathogenicity of Colletotrichum fructicola on Camellia oleifera

Camellia oleifera is the native economic edible oil tree in China and anthracnose occurs commonly in its producing areas. We previously found that ubiquitin-like CfAtg8-related autophagy is essential for the pathogenicity of Colletotrichum fructicola, the major pathogen of anthracnose on C. oleifera. The aim of this study is to further elucidate the roles of autophagy in the pathogenesis of C. fructicola. Based on BLAST_P analysis and reverse genetics, we identified the central hub of another ubiquitin-like system, CfAtg5, and characterized its functions in C. fructicola. As suspected, we found that CfAtg5 is required for autophagy, both in MM-N and H2O2 induction conditions. Targeted gene deletion of CfATG5 revealed that CfAtg5 is involved in growth and conidiation. We further found that the ∆Cfatg5 mutant is defective in appressorium formation and in responses to cell wall integrity and oxidative stresses. The combined effects, together with the abolished autophagy, result in the pathogenicity defects of the ∆Cfatg5 mutant. Our study further illustrates the importance of normal autophagy in the physiology and pathogenicity of C. fructicola, and offers a potential target in the development of new anthracnose control strategies.

E3 ubiquitin ligase PbrATL18 is a positive factor in pear resistance to drought and Colletotrichum fructicola infection

The Arabidopsis Tóxicos en Levadura (ATL) protein is a subfamily of the E3 ubiquitin ligases, which exists widely in plants and is extensively involved in plant growth and development. Although the ATL family has been identified in other species, such as Arabidopsis, Oryza sativa, and grapevine, few reports on pear ATL gene families have been reported. In this study, 92 PbrATL genes were identified and analyzed from the Pyrus breschneideri genome. Motif analysis and phylogenetic tree generation divided them into nine subgroups, and chromosome localization analysis showed that the 92 PbrATL genes were distributed in 16 of 17 pear chromosomes. Transcriptome data and quantitative real-time polymerase chain reaction (qRT-PCR) experiments demonstrated that PbrATL18, PbrATL41, and PbrATL88 were involved in both pear drought resistance and Colletotrichum fructicola infection. In addition, Arabidopsis thaliana overexpressing PbrATL18 showed greater resistance to drought stress than the wild type (WT), and PbrATL18-silenced pear seedlings showed greater sensitivity to drought and C. fructicola infection than the controls. PbrATL18 regulated plant resistance by regulating chitinase (CHI), phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activities. This study provided a reference for further exploring the functions of the PbrATL gene in drought resistance and C. fructicola infection.

Colletotrichum species associated with fig (Ficus carica L.) in Malaysia

Fig (Ficus carica L.) is one of the oldest fruits that is extensively cultivated worldwide. It has gained a lot of interest among growers due to its unique characteristics, nutritional values, and medicinal properties. Like other fruit crops, each part of the plant is susceptible to fungal diseases that affect fruit production and its quality. Samplings conducted from September 2018 to March 2019 showed that fig plants have been infected by leaf blight, stem canker, and anthracnose fruit rot. The present study aims to determine the causal pathogens of those diseases using morphological, molecular, and pathogenicity analyses. A total of 28 blighted leaves, 11 cankered stems, and 18 anthracnose-rotted fruits of F. carica were collected from different nurseries in several states in Malaysia. The results of morphological characterization, DNA sequences, and phylogenetic analysis of internal transcribed spacer (ITS), β-tubulin (tub2), and glyceraldehyde-3-phosphate dehydrogenase (gapdh) revealed that, of the 39 fungal isolates obtained, they belong to Colletotrichum fructicola (one isolate), C. siamense (30 isolates), and C. truncatum (eight isolates). From the 30 isolates of C. siamense, 10 isolates were obtained from leaves, eight isolates from stems, and 12 isolates from fruits, whereas eight isolates of C. truncatum, and one isolate of C. fructicola were obtained from leaves. The pathogenicity tests conducted on F. carica confirmed that C. fructicola, C. siamense, and C. truncatum were the causal pathogens of leaf blight, while stem canker and anthracnose fruit rot were caused by C. siamense. The present study highlighted three different Colletotrichum species namely C. fructicola, C. siamense, and C. truncatum associated with diseased figs (F. carica) in Malaysia.

Post-harvest Anthracnose of Carambola (Averrhoa carambola) Caused by Colletotrichum fructicola in China.

Carambola (star fruit), a popular fruit of Averrhoa carambola in many parts of the world, is considered to have many beneficial nutritional and medicinal effects (Lakmal, K., et al,2021). In March 2020, anthracnose disease was observed on carambola (about 15% of the fruit showed similar symptoms) in multiple local agricultural markets (113°36'E, 23°11'N) of the Yuancun district in Guangzhou, China. Initial symptoms of infected fruit samples appeared as water-soaked, brown lesions. As the disease progressed, numerous acervuli appeared on fruit surfaces. Salmon-colored spore masses were observed on some fruit. To isolate and identify the pathogen, small pieces (3-5 mm2) were excised from the lesion margins of the fruit, which were surface disinfested by 1% NaOCl (60 s), 70% ethanol (30 s) and then washed twice with sterile distilled water. After surface disinfestation, the tissues were cultured on potato dextrose agar (PDA). Pure cultures were obtained by transferring hyphal tips onto fresh PDA. Fungal isolates (YT-5/6/9) were obtained and the strain YT-5 was selected for further study. The colony of strain YT-5 grown on PDA for 7 days appeared to be cottony, white to pale gray with the presence of multiple masses of conidia. Conidia 13.5-20 × 4.8-6.5 μm (n = 50), hyaline, aseptate, straight and cylindrical with rounded ends. Perithecia were thick-walled and globose with a prominent, narrow neck. Asci 37.1-60.2 × 7.1-11.3 μm (n = 25), 4-8 spored, clavate to cymbiform. Ascospores 7.1-17.2 × 4.5-6.5 μm (n = 35), hyaline, large guttulate at the center, slightly curved with rounded ends. Based on the morphological characteristics, the strain was identified as Colletotrichum fructicola (Prihastuti et al. 2009). The molecular identity of the isolates was confirmed by sequencing the internal transcribed spacer (ITS) rDNA region, chitin synthase (CHS-1), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-tubulin (TUB2) genes (Prihastuti et al. 2009, Weir et al. 2012). BLASTN analysis of isolate YT-5 sequences, which were deposited in GenBank (ON428449, ON462353, ON886225, ON886224, ON462354) showed 100% identity with those of Colletotrichum fructicola (MW513778.1, MT918417.1, MW426526.1, MN525875.1, MT941526.1), respectively. A phylogenetic tree analysis based on the concatenated sequences confirmed the isolate YT-5 as C. fructicola. Pathogenicity tests were conducted on fresh fruit of carambola with the isolate YT-5. Healthy fruit was surface disinfested and inoculated with 5 mm mycelial discs of the strain YT-5 after being wounded with a needle or unwounded. Control fruit was inoculated with sterilized PDA plugs. All inoculated fruit was incubated at 26°C for 10 days post inoculation. Control fruit remained asymptomatic, whereas inoculated fruit developed symptomatic at the point of inoculation. The pathogenicity test was performed in duplicate. The pathogenic isolate of C. fructicola was successfully re-isolated on PDA from the symptomatic fruit, thus confirming Koch's postulates. C. fructicola has also been reported as a dominant and aggressive causal agent of anthracnose on sandy pear and avocado in China (Zhang et al. 2015; Li et al. 2022). To our knowledge, this is the first study to isolate and characterize C. fructicola causing carambola anthracnose and evaluate its pathogenicity in China, which will provide a better strategy for accurate diagnosis and effective management of anthracnose disease on carambola.

First Report of Colletotrichum fructicola Causing anthracnose on macadamia in China.

Macadamia (Macadamia ternifolia Maiden and Betche) is an important commercial crop in the world and has the reputation of being the king of nuts (Liu et al. 2019). In August 2020, symptoms of anthracnose appeared on leaves of macadamia in Chongzuo, Guangxi Province, China, with an incidence of 15-20%. The disease developed from the edge of leaf. Initially, the disease symptoms on leaves were faded green spots, light yellow. After expanding and linking together, the leaves appeared brown or black irregular spots, and the edges of diseased leaves dried up and formed large necrosis, eventually leading to defoliation. A large number of orange-yellow spots (acervuli) developed on the diseased parts. Under high humidity conditions, the diseased part was grayish-brown or black, and a large number of yellowish-brown conidia were produced on the leaf surface (Fig.1 A-E). Ten symptomatic leaves were collected and washed with distilled water. Twelve lesion marginal tissues were sterilized with 75% ethanol (V/V) for 30 s and 1% NaOCl for 1min and rinsed with sterile distilled water, plated on potato dextrose agar (PDA) and incubated at 28°C under light. After 3 days, the incubated samples all produced similar cultural morphology. One isolate named GXMC2 as a representative was selected for following study. The colony by single-spore purification on PDA were grayish green with a white outer ring and cottony on surface, pale blackish green in reverse side (Fig.1 F). Conidia with oil droplets were solitary, cylindrical, transparent and measuring 13.78 to 19.25 μm (average 16.90 μm) × 5.14 to 7.33 μm (average 6.23 μm) (n=100) (Fig.1 G). Appressoria were brown to dark brown, with different shapes such as ovoid, elliptical or irregular, some with lobes. The average size was 7.89 to 13.25 μm (average 10.64 μm) × 5.76 to 9.02 μm (average 7.86 μm) (n=100) (Fig.1 H). No setae were found. The isolate was identified as Colletotrichum fructicola on the basis of the morphology of the colonies, conidia and appressoria (Park et al. 2018). The six target gene sequences, including internal transcribed spacer (ITS), β-tubulin (TUB), actin (ACT), histone3 (HIS3), chitin synthase A (CHS), and glyceraldehydes-3-phosphate dehydrogenase (GAPHD) (Qiu et al. 2020), were selected for PCR amplification. The resulting sequences were deposited in GenBank under accession numbers MZ821661, MZ821660, MZ821662, MZ821663, MZ821664 and MZ821665 respectively. Phylogenetic analysis of the concatenated sequences were performed with MEGA 7.0 software. The isolate was grouped in the same clade as other C. fructicola (Fig.2). In May 2022, Inoculation was conducted in the field. Four-year-old macadamia leaves were disinfected with 75% alcohol. The conidial suspension was sprayed on 5 unwounded healthy leaves, and 5 leaves sprayed with sterile distilled water served as control. The experiment was replicated 3 times, with each replicate containing 5 leaves. The average daily temperature and average daily relative humidity in the field were 30°C and 62%, respectively. After 2 days, yellow-brown spots appeared on the inoculated leaves and expanded outward. After 4 days, the diseased areas were dark brown, and the controls remained asymptomatic. The same fungal pathogen was reisolated and purified from inoculated leaves and the identity was confirmed by morphological characterization and molecular analysis, confirming Koch's postulates (Fig.1 I-J). In China, C. fructicola has been reported on Passiflora edulis Sims, Brassica parachinensis, Illicium verum, Peucedanum praeruptorum, etc. (Li et al. 2021; Yu et al. 2022; Zhao et al. 2021; Ma et al. 2020). To our knowledge, this is the first report of anthracnose of macadamia caused by C. fructicola in China. This study provides the basis for further research on this disease.

First Report of Colletotrichum fructicola Causing Anthracnose on Rosa chinensis in China.

China rose (Rosa chinensis Jacq.) is a popular ornamental plant grown widely in China. In September 2021, a serious leaf spot disease was observed on R. chinensis in Rose plantation of Nanyang Academy of Agricultural Sciences in Nanyang (112°25'41″N, 32°54'28″E), Henan Province, causing severe defoliation of infected plants with a foliar disease incidence of 50 to 70% (n = 100). The early symptoms were irregular brown specks on the leaves, mostly at the tip and margin of the leaves. Then the specks gradually expanded into round amorphous and became dark brown, eventually leading to large irregular or circular lesions. Twenty symptomatic samples were collected from several individual plants, and the junction areas between infected and healthy tissues were cut into 3×3 mm pieces. These tissues were sterilized in 75% ethanol for 30 seconds and 1% HgCl solution for 3 min, rinsed thrice in sterile water, and placed on potato dextrose agar (PDA) plates, incubated at 25°C for 3 days. The edges of the colony were cut and transferred to new PDA plates for purification. These isolates were isolated from the original diseased leaves and showed similar phenotypes in morphological characters. Three representative purified strains (YJY20, YJY21, and YJY30) were used for further study. Colonies were villiform, initially white, later turning gray and greyish-green. Conidia were unitunicate, clavate, and averaged 17.36 (11.61 to 22.12) - 5.29 (3.92 to 7.04) µm in diameter (n=100). The characteristics were close to those of Colletotrichum spp. (Weir et al. 2012). The genomic DNA was extracted, and the rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GADPH), calmodulin genes (CAL), actin genes (ACT), chitin synthase 1 genes (CHS-1), manganesesuperoxide dismutase (SOD2), and β-tubulin 2 genes (TUB2) were amplified from genomic DNA by primers ITS1/ITS4, GDF/GDR, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-345R, SODglo2-F/SODglo2-R, and Bt2a/Bt2b, respectively (Weir et al. 2012). Sequences were submitted to GenBank with accession numbers OP535983, OP535993, OP535994(ITS), OP554748, OP546349, OP546350(GAPDH), OP546351-OP546353(CAL), OP546354-OP546356(ACT), OP554742-OP554744(CHS-1), OP554745-OP554747(SOD2), and OP554749-OP554751(TUB2). BLASTn analyses of ITS, GAPDH, CAL, ACT, CHS-1, SDO2 and TUB2 sequences exhibited 99.62%, 98.40%, 99.72%-99.86%, 96.85%-96.86%, 99.26%-100%, 100% and 99.33% similarity to the sequences of Colletotrichum fructicola strain ICMP 18581, respectively in GenBank. These morphological features and molecular identification indicated that the pathogen possessed identical characteristics as C. fructicola (Weir et al. 2012). Pathogenicity was tested through in vivo experiments. Six 1-year-old intact plants were used per isolate. The test leaves of plants were gently scratched with a sterilized needle. Conidial suspension of the pathogen strains were inoculated on the wounded leaves at a concentration of 107 conidial/mL. The control leaves were inoculated with distilled water. The inoculated plants were placed in greenhouse at 28℃ and 90% humidity. After 3-6 days，anthracnose-like symptoms were observed on inoculated leaves of five plants while the control plants remained healthy. The strains of C. fructicola were reisolated from the symptomatic inoculated leaves, confirming Koch's postulates. To our knowledge, this is the first report of C. fructicola causing anthracnose on Rosa chinensis in China. C. fructicola has been reported to affect numerous plants worldwide, including grape, citrus, apple, cassava, mango （Qili Li et al. 2019）, and tea-oil tree (X. G. Chen et al. 2022), among others (Oliveira et al. 2018). This identification research will facilitate subsequent assistance with disease control and field management of plants.

Genome-Wide Identification and Expression Analysis of Rosa roxburghii Autophagy-Related Genes in Response to Top-Rot Disease.

Autophagy is a highly conserved process in eukaryotes that degrades and recycles damaged cells in plants and is involved in plant growth, development, senescence, and resistance to external stress. Top-rot disease (TRD) in Rosa roxburghii fruits caused by Colletotrichum fructicola often leads to huge yield losses. However, little information is available about the autophagy underlying the defense response to TRD. Here, we identified a total of 40 R. roxburghii autophagy-related genes (RrATGs), which were highly homologous to Arabidopsis thaliana ATGs. Transcriptomic data show that RrATGs were involved in the development and ripening processes of R. roxburghii fruits. Gene expression patterns in fruits with different degrees of TRD occurrence suggest that several members of the RrATGs family responded to TRD, of which RrATG18e was significantly up-regulated at the initial infection stage of C. fructicola. Furthermore, exogenous calcium (Ca2+) significantly promoted the mRNA accumulation of RrATG18e and fruit resistance to TRD, suggesting that this gene might be involved in the calcium-mediated TRD defense response. This study provided a better understanding of R. roxburghii autophagy-related genes and their potential roles in disease resistance.

Identification of Camellia oleifera WRKY transcription factor genes and functional characterization of CoWRKY78.

Camellia oleifera Abel is a highly valued woody edible oil tree, which is endemic to China. It has great economic value because C. oleifera seed oil contains a highproportionof polyunsaturated fatty acids. C. oleifera anthracnose caused by Colletotrichum fructicola, poses a serious threat to C. oleifera growthandyield and causes the benefit of the C. oleifera industry to suffer directly. The WRKY transcription factor family members have been widely characterized as vital regulators in plant responsetopathogeninfection. Until now, the number, type and biological function of C. oleifera WRKY genes are remains unknown. Here, we identified 90C. oleifera WRKY members, which were distributed across 15 chromosomes. C. oleifera WRKY gene expansion was mainly attributedto segmental duplication. We performed transcriptomic analyses to verify the expression patterns of CoWRKYs between anthracnose-resistant and -susceptible cultivars of C. oleifera. These results demonstrated that multiple candidate CoWRKYs can be induced by anthracnose and provide useful clues for their functional studies. CoWRKY78, an anthracnose-induced WRKY gene, was isolated from C. oleifera. It was significantly down-regulated in anthracnose-resistant cultivars. Overexpression of CoWRKY78 in tobacco markedly reduced resistancetoanthracnose than WT plants, as evidenced by more cell death, higher malonaldehyde content and reactive oxygen species (ROS), but lower activities of superoxide dismutase (SOD), peroxidase (POD), as well as phenylalanine ammonia-lyase (PAL). Furthermore, the expression of multiple stress-related genes, which are associated with ROS-homeostasis (NtSOD and NtPOD), pathogen challenge (NtPAL), and pathogendefense (NtPR1, NtNPR1, and NtPDF1.2) were altered in the CoWRKY78-overexpressing plants. These findings increase our understanding of the CoWRKY genes and lay the foundation for the exploration of anthracnose resistance mechanisms and expedite the breeding ofanthracnose-resistant C. oleifera cultivars.

Genome Resources for the Colletotrichum gloeosporioides Species Complex: 13 Tree Endophytes from the Neotropics and Paleotropics.

Thirteen draft genome assemblies are presented for four Colletotrichum gloeosporioides complex species, namely, Colletotrichum aeschynomenes, Colletotrichum asianum, Colletotrichum fructicola, and Colletotrichum siamense, which were isolated from tropical tree hosts as endophytes.

First Report on Colletotrichum fructicola Causing Anthracnose in Chinese Sorghum and Its Management Using Phytochemicals.

Sorghum bicolor is cultivated worldwide. Leaf spots on sorghum, which lead to leaf lesions and impaired growth, are prevalent and severe in Guizhou Province, Southwest China. In August 2021, new leaf spot symptoms were observed on sorghum plants growing in agricultural fields. We used conventional tissue isolation methods and pathogenicity determination tests. Inoculations of sorghum with isolate 022ZW resulted in brown lesions similar to those observed under field conditions. The original inoculated isolates were reisolated and fulfilled Koch's postulates. Based on the morphological character and phylogenetic analyses of the combined sequences of the internal transcribed spacer (ITS) region and the β-tubulin (TUB2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, we identified the isolated fungus as C. fructicola. This paper is the first to report this fungus-causing disease in sorghum leaves. We studied the sensitivity of the pathogen to various phytochemicals. The sensitivity of C. fructicola to seven phytochemicals was measured using the mycelial growth rate method. Honokiol, magnolol, thymol, and carvacrol displayed good antifungal effects, with EC50 (concentration for 50% of the maximal effect) values of 21.70 ± 0.81, 24.19 ± 0.49, 31.97 ± 0.51, and 31.04 ± 0.891 µg/mL, respectively. We tested the control effect of the seven phytochemicals on the anthracnose caused by C. fructicola: honokiol and magnolol displayed good field efficacy. In this study, we expand the host range of C. fructicola, providing a basis for controlling sorghum leaf diseases caused by C. fructicola.

First Report of Colletotrichum fructicola Causing Fruit Rot and Leaf-Tip Dieback on Pineapple in Northern Thailand.

Pineapple is one of the most economically important fruits in tropical countries, particularly in Thailand. Canned pineapple is currently Thailand's main exported commodity to many countries, including the United States, Russia, Germany, Poland, and Japan. Fungal diseases are considered a permanent threat to fruits in the pre- and post-harvest stages, leading to considerable economic losses. Fungal disease is one of the primary causes of massive yield losses in pineapples around the world. Colletotrichum species are the most common fungal pathogens affecting different tropical fruits. Although there are many reports regarding Colletotrichum species associated with pineapple, they do not have molecular data to confirm species identification. However, the occurrence of Colletotrichum species on pineapple has not been reported in Thailand so far. In this study, we isolated and identified Colletotrichum fructicola on pineapple in northern Thailand and have proven its pathogenicity to the host. This is the first report of the occurrence of Colletotrichum in pineapple, based on morpho-molecular approaches.