Molecular, morphophysiological, and pathogenic characterization of Colletotrichum isolates from strawberry plants in Santa Catarina, Brazil.

Endophytic Fusarium isolates from Ceratozamia mirandae enhance tomato growth, suppress pathogenic fungi, and induce protection against Botrytis cinerea.

ABSTRACTFrangipani (Plumeria spp.: Apocynaceae) is a small ornamental tree native to Mexico, susceptible to significant diseases such as anthracnose and foliar blight in hot, dry environments. Understanding the diversity of pathogens responsible for these diseases is crucial for developing effective disease management strategies. This study aimed to identify the pathogens responsible for these diseases. In February 2020 and 2021, symptoms of anthracnose and foliar blight were observed on frangipani plants in recreational areas of Morelos and Sinaloa, Mexico. Isolations from diseased leaf tissue resulted in the recovery of 12 Colletotrichum isolates from P. rubra and 18 Alternaria isolates from P. obtusa. After morphotype grouping, a representative isolate from each group was identified by morphology and phylogenetic reconstruction (ITS, act, gapdh, rpb2 and tef1) as Colletotrichum karstii, Colletotrichum siamense, Alternaria destruens and Alternaria burnsii. For pathogenicity testing, a conidial suspension was sprayed onto the leaves of 90‐day‐old plants, while control plants were sprayed with sterile distilled water. All inoculated fungi were pathogenic, reproducing the characteristic symptoms of the disease, while control plants remained symptomless. The fungi recovered from symptomatic plants were morphologically identical to the inoculated fungi, fulfilling Koch's postulates. This is the first report of A. burnsii and A. destruens causing foliar blight in P. obtusa, and C. siamense and C. karstii causing anthracnose in P. rubra. These findings contribute valuable insights for developing effective disease management strategies.

Here, we isolated two novel mycoviruses coinfecting the fugus Colletotrichum karstii, which were designated as "Colletotrichum karstii mitovirus 1" (CkMV1) and "Colletotrichum karstii mitovirus 2" (CkMV2). The length of the complete genome of CkMV1 is 2,441 nucleotides, and that of CkMV2 is 2,391 nucleotides. Both contain a single open reading frame (ORF) that encodes an RNA-dependent RNA polymerase (RdRp). BLAST searches showed that the amino acids (aa) sequences of CkMV1 and CkMV2 had the highest amino acid sequence identity of 35.41% and 52.37% to the RdRp of Pleurotus pulmonarius duamitovirus 1 and Fusarium oxysporum f. sp. cubense mitovirus 4, respectively. Phylogenetic analysis based on RdRp sequences revealed that CkMV1 grouped with members of the genus Duamitovirus and that CkMV2 grouped with members of the genus Unuamitovirus, both within the family Mitoviridae.

The perennial herbaceous vine Stephania epigaea Lo, known as Di Burong in Chinese, is generally used as an anti-inflammatory and analgesic drug, as well as an ornamental plant (Dong et al., 2018). In May 2024, symptoms resembling anthracnose were observed on S. epigaea plants in an herbal nursery (33 m2, approximately 400 plants) in Kunming, Yunnan Province (24°33' 59.21" N, 99°55' 43.71" E), China, with an incidence rate of 75%. The disease initially occurred as small, round brown spots on the leaf margins, which later expanded into irregular lesions. The centers of these lesions were dark brown with yellow edges. To isolate and identify the pathogen, 0.5 × 0.5 mm sections of diseased leaf tissue were sterilized by dipping them in 75% ethanol for 30 s, then in 1% NaCIO for 180 s, followed by three rinses in sterile water. The samples were then cultured on potato dextrose agar (PDA) in the dark at 28 °C for 3 days. The fungal isolates were purified using the single-spore purification method, and 10 isolates with similar morphological characteristics were obtained. The colonies on PDA were initially white, changing to a grayish-white color over time. The ascomata were dark brown, spherical, and either on the surface or partially immersed in the PDA. The conidiophores were smooth, septate, branched, and hyaline to light brown. The conidia were cylindrical, aseptate, hyaline, and smooth, with dimensions of 10.0 to 16.7 × 5.2 to 6.9 μm (average 13.5 × 6.0 μm, n = 30). A 24-hour slide culture (Cai et al., 2009) of the conidia suspension revealed solitary appressoria that were light to dark brown, nearly spherical to oval, and smooth-walled to undulate, with dimensions of 6.8 to 10.5 × 4.7 to 10.0 μm (average 8.7 × 6.7 μm, n = 30). To further characterize the pathogen, isolate SeF03 was randomly selected for further testing. Total genomic DNA was extracted using the cetyl trimethylammonium bromide method, and the nuclear ribosomal internal transcribed spacers (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone 3 (HIS3), actin (ACT), and calmodulin (CAL) genes were amplified using the primer pairs ITS1/ITS4 (White et al., 1990), gpd1/gpd2 (Berbee et al., 2019), CYLH3F/CYLH3R (Crous et al., 2006), ACT-512F/ACT-783R (Carbone and Kohn, 1999), and CL1C/CL2C (Weir et al., 2012), respectively. BLASTn homology search showed that the ITS (PQ963009), GAPDH (PV021394), HIS3 (PV021395), ACT (PV021396), and CAL (PV021397) sequences of SeF03 were 100, 98.81, 100, 98.15, and 100% identical to Colletotrichum karstii strain CBS 128500 (JQ005202, JQ005289, JQ005463, JQ005550, and JQ005723), respectively. A phylogenetic tree of Colletotrichum species was built based on concatenated nucleotide sequences of ITS, GAPDH, HIS3, ACT, and CAL using the maximum likelihood method with the Tamura-Nei model. SeF03 and C. karstii clustered on the same branch. Morphological and molecular characteristics also confirmed that SeF03 was identical to C. karstii. Pathogenicity was evaluated through foliar spray inoculation with a conidial suspension (1 × 10⁶ conidia/mL) applied until runoff, using sterile water-treated plants as controls. All sprayed plants were placed in an incubator at a constant temperature of 28 ℃ with alternating light and dark periods (L/D = 12 h/12 h, 80% humidity). Each treatment consisted of 3 plants, with 2 replicates. After 15 days, obvious brown spots appeared on leaves treated with the SeF03 spore solution, while the control plants sprayed with sterile water remained asymptomatic. The fungal strains isolated from the symptomatic leaves shared the same morphological characteristics as SeF03. C. karstii has been reported to cause anthracnose on Fatsia japonica, Dalbergia odorifera, and Piper nigrum. To our knowledge, this is the first report of anthracnose caused by C. karstii on S. epigaea in China. The disease significantly threatens S. epigaea's ornamental value, requiring urgent and targeted control measures.

Codiaeum variegatum is an ornamental plant widely cultivated in various regions of the world, valued for the coloration and diversity of its leaves. In July 2022, reddish-brown lesions were observed on the leaves of plants cultivated in a residence in the state of Rio Grande do Sul, Brazil. In view of this, the aim of this study was to isolate, characterize, and identify the causal agent of anthracnose in C. variegatum through morphological and molecular traits. Pure cultures were obtained from the lesions in potato dextrose agar (PDA). To fulfill Koch’s postulates, disks of agar containing fungal structures were placed on healthy C. variegatum leaves. After the incubation period under controlled conditions, red spots were observed on the inoculated areas, similar to the original lesions. The isolates were morphologically characterized and molecularly identified using the ITS (Internal Transcribed Spacer), Calmodulin (CAL), and Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) gene regions. The morphological and sequence analysis confirmed the species Colletotrichum karstii and Colletotrichum gigasporium as the causal pathogens of anthracnose in C. variegatum. The sequences of the ITS, CAL, and GAPDH genes from the isolates were deposited in GenBank, and a culture of each pathogen was deposited in the Herbarium of the Department of Biology at the Universidade Federal de Santa Maria. The pathogens were registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge. This is the first report of C. karstii and C. gigasporium causing anthracnose in C. variegatum in Brazil.

Danshen (Salvia miltiorrhiza Bunge), a member of the genus Salvia within the Lamiaceae family, holds significant economic and medicinal value. Regrettably, the emergence of a novel leaf anthracnose in 2020 has significantly impacted its cultivation, leading to decreased yield and compromised quality. This newly identified pathogen was meticulously isolated from affected leaves, employing meticulous single conidia isolation techniques. Subsequent confirmation of pathogenicity was achieved through strict adherence of Koch's postulates. To ensure precise identification, morphological characteristics were supplemented with tandem sequence analysis targeting the rDNA internal transcribed spacer (ITS), β-tubulin (TUB), and histone (His3) regions. Combining molecular biology techniques with morphological observation and Koch's postulates, the pathogen was conclusively identified as Colletotrichum karstii. Further investigations focused on understanding the environmental factors influencing the mycelial growth and sporulation of the pathogen. The optimum temperature for the growth of C.karstii is 25°C, the suitable light conditions are 12h light/12h dark or 24h dark, and the suitable pH is 5 to 9. Utilizing BIOLOG phenotypic analysis technique, the metabolic utilization of carbon and nitrogen sources by the pathogen was assessed across different temperatures (20°C, 25°C, and 30°C). Results indicated the highest utilization rates at 25°C, particularly for arbutin and L-tryptophan. Lastly, the efficacy of 15 chemical fungicides and six botanical fungiticide against C. karstii was evaluated in vitro, revealing fluazinam as the most potent inhibitor against mycelial growth with EC50 of 0.0725 mg/mL for mycelium and 0.0378 mg/mL for spore germination, respectively. The 1 % osthole emulsion in water was found to have the strongest inhibitory effect on the growth of mycelium, with an EC50 value of 4.8984 µg/mL. Spore germination was most strongly inhibited by the 80 % ethylicin EC, which had an EC50 value of 0.5541 µg/mL. This study represents the first documentation of C. karstii as a causative agent of anthrax in Danshen, underscoring the significance of these findings for agricultural management and disease control strategies.

Epimedium sagittatum (Sieb.et Zucc.) Maxim, belonging to the family Berberidaceae and the genus Epimedium, is effective in dispersing wind and cold, tonifying the kidneys and bones, and strengthening muscles and bones (Song et al. 2024). In June 2023, a new leaf disease appeared on 33.35 hm2 of E. sagittatum cultivation in Xinning County, Shaoyang City, Hunan Province, China (111°43'00″N, 27°26'40″E), with symptoms appearing on about 50% of the cultivation area. Initially lesions appear on the leaf tips, then the spots gradually widen and become yellowish-white and dry, with a dark yellow halo around the periphery. We collected ten infected leaves from different growing areas and rinsed them with running sterile water. To isolate the pathogen, small infected parts cut from the leaves were surface disinfected with 3% hydrogen peroxide for 30 s and then treated with 75% alcohol for 45 s. Finally, the diseased parts were rinsed five times with sterile water and incubated on water agar (WA) plates at 28°C for 3-5 days. After mycelial growth was observed, the hyphae were transferred to potato dextrose agar (PDA) plates and incubated for 3-5 days at 28°C in the dark. Finally, eight isolates were obtained, and six of them belonged to Colletotrichum genus basedontheir morphological characterisation on PDA plates (isolation frequency 75%). Three representative strains (YYH5, YYH6 and YYH7) were selected for further study. The colonies were initially round with white aerial and later grey-black to white on the back. Under the microscope the conidia were short and cylindrical. They had rounded ends and measured 12.936 to 15.894 × 6.119 to 8.137μm (n = 100). To further confirm the identity of the isolates, five gene regions of the isolates, including internal transcribed spacer (ITS), actin (ACT), chitin synthase (CHS), calmodulin (CAL) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were amplified and sequenced using the universal primers ITS4/ITS5, ACT-512F/ACT-783R, CHS-79F/CHS-345R, CL1C/CL2C and GDF/GDR respectively (Weir et al. 2012). Sequences were submitted to GenBank (ITS: PP898147, PP898149, PP898150; ACT: PP925578, PP925579, PP925585; CHS: PP925576, PP925577, PP925584; CAL: PP925580, PP925581, PP925586; GAPDH: PP925582, PP925583, PP925587). Analysis of the constructed five-gene joint phylogenetic tree showed that these three isolates were significantly clustered with Colletotrichum karstii. Pathogenicity was tested using three representative strains. The epidermis of young E. sagittatum leaves was punctured with a sterile needle and 6 × 6 mm mycelial blocks cultured on PDA for 7 days were placed on the wound. Controls were treated in the same way except that sterile PDA blocks were used. There were three replicates for each treatment. All plants used in the experiment were kept in a constant climate chamber at 28°C. The photoperiod was 12 hours and the relative humidity of the chamber was maintained at 80%. Three days later, lesions appeared at the inoculation sites on the plants. Symptoms worsened after five days. All strains re-isolated from diseased spots had the same colony characteristics as the representative strain and were confirmed as C. karstii by DNA sequencing. Therefore, Koch's postulates is fulfilled. C. karstii has been shown to cause anthracnose on Fragaria x ananassa Duch (Soares et al. 2021), Piper nigrum L. (Lin et al. 2022) and Glycine max (Luo et al. 2024). To our knowledge, this is the first report of C. karstii causing anthracnose on E. sagittatum. This research will lay the foundations for future control of the disease.

The present study aimed to evaluate the influence of the circadian rhythm and hydrodistillation time on the content and chemical composition of the essential oils (EOs) from the leaves of Callistemon viminalis, as well as their antifungal activity against Colletotrichum gloeosporioides, Colletotrichum karstii and Botrytis cinerea. The EOs from C. viminalis leaves showed yields ranging from 0.54 to 1.67%, with eucalyptol identified as the predominant component in all samples. During the circadian rhythm, samples collected at different times exhibited variations in chemical composition. The EOs from leaves collected at 6 a.m. and 6 p.m. showed higher antifungal potential against B. cinerea, with a 100% inhibition of mycelial growth (500 µL mL-1), while samples from 6 a.m. and 12 p.m. showed effects against C. gloeosporioides with inhibition percentages of 52.3 and 55.9%, respectively (500 µL mL-1). The 6 a.m. sample showed the best inhibition potential against C. karstii, with an inhibition percentage of 68.9% at a concentration of 600 µL mL-1.

Abstract: In June 2022, anthracnose was detected on the leaves of Annona muricata L. (soursop) in Ibirapitanga, Bahia, Brazil. To identify the pathogens, diseased leaves were surface sterilized and cultured on potato dextrose agar, resulting in the selection of two isolates, IBIRA-P5R2 and IBIRA-P2F1R2, which were used for further analysis. IBIRA-P5R2 exhibited gray to white mycelium with dark conidiomata, cylindrical conidia, and dark appressoria. In contrast, IBIRA-P2F1R2 exhibited dense, pale gray mycelium with distinct conidial and ascospore characteristics. Genetic analysis involved sequencing three regions: Internal Transcribed Spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT), with results deposited in GenBank. BLAST analysis revealed that IBIRA-P5R2’s sequences were 95.45% similar to Colletotrichum karstii, while IBIRA-P2F1R2’s sequences were 99.06% identical to . Colletotrichum fructicolaPathogenicity tests confirmed that both isolates caused anthracnose symptoms in soursop seedlings, with dark spots developing on leaves after three days. The pathogens were reisolated from infected leaves, confirming the same cultural and morphological traits as the original isolates. This study marks the firstrecorded occurrence of C. karstii and C. fructicola causing anthracnose on soursop in Bahia, Brazil. The findings aim to inform soursop farmers about this emerging disease, highlighting the need for tailored integrated management strategies to address different plant pathogens effectively.

Endophytic fungi are increasingly being recognized for their diverse metabolites that may exhibit antimicrobial properties. In our study, we isolated seven endophytic fungal strains from Calea pinnatifida, which were identified as Hypomontagnella barbarensis, Neopestalotiopsis clavispora, Nigrospora sacchari-officinarum, Annulohypoxylon moriforme, Colletotrichum siamense, and Colletotrichum karstii (with two isolates from the same species). Furthermore, the antimicrobial activity of the extracts was assessed, revealing that the extract from Hypomontagnella barbarensis demonstrated activity against Staphylococcus aureus. Further investigation of secondary metabolites, employing UHPLC-HR-ESI-MS/MS in combination with molecular networking, facilitated annotation of the nine compounds. Of these, five were identified based on matches with the GNPS spectral library, and four were predicted based on the molecular network. Notably, from the extract of Hypomontagnella barbarensis, two pairs of diastereoisomeric acyl-tetronic acid derivatives were isolated and characterized using MS and NMR spectroscopy. This study highlights the potential of endophytic fungi as a valuable source of novel antimicrobial agents.

Soybean (Glycine max) is a significant grain and oil crop. Among the various challenges faced by soybean cultivation, anthracnose stands out as one of the most prevalent diseases. In June 2023, anthracnose symptoms on leaves characterized by irregular disease spots featuring gray-white centers and brown edges, along with many small black dots on their surface, were observed in a 20-hectare soybean (variety "Liu Yuehuang") field located in Luodian County (25°40'20″ N, 106°53'50″ E, 575 m), Guizhou Province, China. Around 30% of the 300 soybean plants examined were symptomatic, and a total of ten leaves were collected. Fragments (5×5 mm) from the edge of disease spots were sheared and surface-sterilized with 3% sodium hypochlorite and 75% ethanol for 60 s and 30 s, respectively. They were then flushed twice with sterile water, dried using sterile filter papers, finally placed on potato dextrose agar (PDA) and incubated at 28°C for two days. In total, 11 isolates with identical morphological characteristics were obtained. The colonies grown with white aerial mycelia on their surface; conidia were cylindrical, both ends are rounded, aseptate, hyaline, 11.0-14.0 (12.5) × 4.5-6.0 (5.0) μm (n = 30); appressoria were nearly ovoid, brown to black, 8.5-10.5 (9.5) × 5.5-7.5 (6.0) μm (n = 30). The morphological characteristics closely resembled the description of C. karstii (Damm et al., 2012). To further identify the isolates, chitin synthase (CHS-1), actin (ACT), beta-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the internal transcribed spacer (ITS) loci were amplified by using CHS-79F/CHS-345R, ACT-512F/ACT-783R (Carbone and Kohn, 1999), Bt2F/Bt2R (Woudenberg et al., 2009), GDF/GDR (Guerber et al., 2003) and ITS1/ITS4 (White et al., 1990) PCR primers, respectively. The BLAST results showed that the sequences of two representative strains, LD 2023048-1 and LD 2023048-2, were highly similar to those of strain C. karstii CGMCC3.14194 (ITS: OR342620 (99%) and OR342621 (99%) with HM585409, ACT: OR412337 (97%) and OR423341 (100%) with HM581995, CHS-1: OR423344 (99%,) and OR423345 (100%) with HM582023, GAPDH: OR423348 (98%) and OR423349 (98%) with HM585391, and TUB: OR423352 (99%) and OR423353 (99%) with HM585428). The phylogenetic tree combined five sequences showed that the two strains clustered into a branch of C. karstii CGMCC3.14194 with high support values. Thirty-day-old soybean plants (n = 10) (variety Liu Yuehuang) were separately sprayed with 1 × 105 spore suspensions/mL of the two strains by spray method, and plants sprayed with sterile distilled water were used as the negative control (n = 5). All the plants were then covered with plastic bags and cultured in the greenhouse (28℃, 80% humidity, 12 h light dark cycle). After ten days of inoculation, plants inoculated with C. karstii began to produce typical anthracnose symptoms, while the control remained asymptomatic. The confirmation of the reisolated pathogen as C. karstii was established through a comprehensive analysis of morphology and five sequencing loci. Pathogenicity tests were repeated three times. Anthracnose on soybean is caused by Colletotrichum spp. reported in China including C. truncatum (Hu et al., 2015), C. brevisporum (Shi et al., 2021) and C. fructicola (Xu et al., 2023). As far as we know, this study is the initial report of C. karstii inducing anthracnose on soybean to date, which establishes a fundamental reference for preventing and controlling this disease.

Abstract Up to 32 Colletotrichum species have been reported to be associated with pre‐ or postharvest diseases of citrus globally, while in Australia, six species have been reported to cause citrus leaf and fruit disease. Twig or shoot dieback has recently been observed as an emerging disease in citrus orchards in Western Australia. Colletotrichum species were isolated from diseased twigs showing dieback (withertip) or lesions, with or without gummosis, collected from 12 varieties of orange, mandarin and lemon. Colletotrichum gloeosporioides sensu stricto, Colletotrichum karstii and Colletotrichum novae‐zelandiae were identified using a polyphasic approach that included multigene phylogenetic analysis using sequences of internal transcribed spacer and intervening 5.8S nrDNA (ITS), glyceraldehyde‐3‐phosphate dehydrogenase (gapdh), β‐tubulin (tub2), actin (act) and histone (his3) for isolates in the boninense species complex, and Apn2–Mat1–2 intergenic spacer and partial mating type (Mat1–2) (ApMat) and glutamine synthetase (gs) for isolates in the gloeosporioides species complex, as well as morphological characteristics. C. gloeosporioides was the most prevalent species associated with twig dieback in Western Australia, while C. novae‐zelandiae was reported for the first time in Australia. Pathogenicity tests on shoot twigs from lemon and orange trees confirmed C. gloeosporioides, C. karstii and C. novae‐zelandiae as the cause of twig dieback, with C. gloeosporioides being the most aggressive species. Knowledge of the species causing twig dieback and their lifestyle will assist the development of integrated control methods.

Diversity of endophytic fungi from medicinal plant Oxalis latifolia and their antimicrobial potential against selected human pathogens

Dalbergia odorifera (Family: Fabaceae) is a national second-grade protected tree in China with high medicinal and economic value (Zhao et al., 2020). In July, 2022, a leaves spot disease on D. odorifera with typical anthracnose symptoms was observed in plantations in Haikou (110.319153°E, 19.072900°N), Dongfang (108.630297°E, 19.103838°N) and Qiongzhong (109.704460°E, 19.088440°N), Hainan Province, China. Disease incidence was 7.5% (n = 50 plants). Early symptoms of infected leaves were small and round dark brown spots, which developed into larger irregular necrotic lesions and leaves withered. Leaf tissues (5×5 mm) at the disease-health junction of spots from 19 leaves were sterilized with 2.5% sodium hypochlorite for 1 min, and rinsed with sterile distilled water three times. These sterilized tissues were placed on potato dextrose agar (PDA) and incubated at 28 ℃ for 5 d. 7 strains of fungi with similar morphology were isolated, and 3 single-hyphal isolates (HHL01, HHL02 and HHL03) from each location were selected for further study. Colonies on PDA were fluffy orange-yellow mycelium. Conidia were aseptate, cylindrical, smooth-walled, straight, hyaline with both ends bluntly rounded, 11.82 to 15.77 × 3.87 to 6.71 μm (n = 100; average = 13.75 × 5.52 μm). Appressoria formed on slides, measured 5.54 to 10.64 × 4.19 to 7.41 μm (n = 30; average = 8.06 × 5.97 μm) were brown to black, elliptical to irregular. For molecular biological identification, the genomic DNA of three isolates was extracted by fungal genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.). The partial sequences of internal transcribed spacer region (ITS; ITS1/ITS4), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; GDF1/GDR1), actin (ACT; ACT512F/ACT783R), β-tubulin (TUB2; TI/Bt2b) and calmodulin (CAL; CL1C/CL2C) were amplified and sequenced by Sangon Biotech (Shanghai) Co., Ltd (Carbone and Kohn, 1999; Weir et al., 2012). The sequences were deposited as GenBank Accession Nos. OR018110-OR018112 (ITS); OR050529-OR050537 (GAPDH, ACT and CAL) and OR192168-OR192170 (TUB). BLASTn results showed these sequences were more than 99% identity with the strain of C. karstii CORCK1 (GenBank Accession Nos. HM585406, HM585387, HM581991, HM585424 and HM582010, respectively). Multi-locus phylogenetic tree of Colletotrichum spp. showed that those three isolates were sister to C. karstii based on the maximum likelihood and bayesian inference methods. To verify pathogenicity, 2 mL spore suspension (1 × 106conidia/ml) of the isolates was sprayed on each leaves of 1-year-old D. odorifera plants, and sterile distilled water was similarly sprayed on other leaves as a negative control. The plants were incubated in a greenhouse under 90% ± 5% RH at 28 °C. Light brown small round necrotic patches developed 3 days after inoculation, while the control was asymptomatic. Photographs were taken on the fifth day after inoculation. The fungi were re-isolated from the diseased leaves and identified by morphological characterization and molecular identification, fulfilling Koch's postulates. C. karstii has been reported causing leaf rot of Carissa grandiflora in Spain (Garcia-Lopez et al., 2021), and anthracnose caused by C.tropicale was reported on D. odorifera (Yi et al., 2023). To our knowledge, this is the first report of Dalbergia odorifera leaf spot disease caused by Colletotrichum karstii. This finding provides an important basis for further research on the control of the disease.

Rhizoctonia solani is a significant pathogen affecting various crops, including tobacco. In this study, a bacterial strain, namely Y246, was isolated from the soil of healthy plants and exhibited high antifungal activity. Based on morphological identification and DNA sequencing, this bacterial strain was identified as Bacillus safensis. The aim of this investigation was to explore the antifungal potential of strain Y246, to test the antifungal stability of Y246 by adjusting different cultivation conditions, and to utilize gas chromatography-mass spectrometry (GC-MS) to predict the volatile compounds related to antifungal activity in Y246. In vitro assays demonstrated that strain Y246 exhibited a high fungal inhibition rate of 76.3%. The fermentation broth and suspension of strain Y246 inhibited the mycelial growth of R. solani by 66.59% and 63.75%, respectively. Interestingly, treatment with volatile compounds derived from the fermentation broth of strain Y246 resulted in abnormal mycelial growth of R. solani. Scanning electron microscopy analysis revealed bent and deformed mycelium structures with a rough surface. Furthermore, the stability of antifungal activity of the fermentation broth of strain Y246 was assessed. Changes in temperature, pH value, and UV irradiation time had minimal impact on the antifungal activity, indicating the stability of the antifungal activity of strain Y246. A GC-MS analysis of the volatile organic compounds (VOCs) produced by strain Y246 identified a total of 34 compounds with inhibitory effects against different fungi. Notably, the strain demonstrated broad-spectrum activity, exhibiting varying degrees of inhibition against seven pathogens (Alternaria alternata, Phomopsis. sp., Gloeosporium musarum, Dwiroopa punicae, Colletotrichum karstii, Botryosphaeria auasmontanum, and Botrytis cinerea). In our extensive experiments, strain Y246 not only exhibited strong inhibition against R. solani but also demonstrated remarkable inhibitory effects on A. alternata-induced tobacco brown spot and kiwifruit black spot, with impressive inhibition rates of 62.96% and 46.23%, respectively. Overall, these findings highlight the significant antifungal activity of B. safensis Y246 against R. solani. In addition, Y246 has an excellent antifungal stability, with an inhibition rate > 30% under different treatments (temperature, pH, UV). The results showed that the VOCs of strain Y246 had a strong inhibitory effect on the colony growth of R. solani, and the volatile substances produced by strain Y246 had an inhibitory effect on R. solani at rate of 70.19%. Based on these results, we can conclude that Y246 inhibits the normal growth of R. solani. These findings can provide valuable insights for developing sustainable agricultural strategies.

Banana Shrub (Michelia figo (Lour.) Spreng.) is widely cultivated in most of southern China (Wu et al, 2008). It can be used to make essential oil and flower tea（Ma et al, 2012; Li et al, 2010）.The first symptoms were observed in Sept. 2020 at a grower's field in Banana shrub seedlings (0.6 ha), Ya'an city (29°30'N, 102°38'E), Hanyuan county. The symptoms re-occurred in May-June of 2021 and became prevalent from August to September. the incidence rate and the disease index were 40% and 22%, respectively. Initially, purplish-brown necrotic lesions appeared at the leaf tip with dark-brown edges. Progressively, necrosis spread, to the middle of the leaves, and the older area turned gray-white. Dark sunken lesions appeared in the necrotic areas and orange conidial masses were visible under humid conditions. Ten isolates were obtained on potato dextrose agar (PDA) from 10 leaf samples using previously described tissue isolation method (Fang et al. 1998). All the 10 isolates exhibited similar morphological characteristics. Grey to white aerial mycelium at the center and in dispersed tufts, with numerous dark conidiomata scattered over the surface, reverse was pale orange with numerous dark flecks corresponding to the ascomata, orange conidial masses were formed from mature conidiomata. Conidia were hyaline, smooth-walled, aseptate, straight, cylindrical, apex round, the contents appearing granular 14.8 to 17.2 × 4.2 to 6.4 μm (average: 16.26 × 4.84 μm, n=30) as Colletotrichum spp. (Damm et al. 2012). For molecular identification, DNA was extracted from a representative isolate HXcjA using a plant genomic DNA extraction kit (Solarbio, Beijing). and the partial sequences of internal transcribed spacer region (ITS, OQ641677), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, OL614009), actin (ACT, OL614007), beta-tubulin (TUB2, OL614011), histone3 (HIS3, OL614010), and calmodulin (CAL, OL614008) were amplified and sequenced using the primer pairs ITS1/ITS4 (White et al. 1990), GDF/GDR (Templeton et al. 1992), ACT-512F/ACT-783R, CAL 228F/CAL 737R (Carbone et al. 1999), TUB1F/Bt2bR, CYLH3F/CYLH3R (Crous et al. 2004), respectively. BLASTn analysis for ITS, GAPDH, CAL, ACT, TUB2 and HIS3 sequences showed ≥99.7% identity to C. Karstii, namely, NR_144790 (532/532 bp), MK963048 (252/252 bp), MK390726 (431/431 bp), MG602039 (761/763 bp), (KJ954424, 294/294 bp), (KJ813519, 389/389 bp), respectively. The fungus was identified as C. karstii based on morphology and a multigene phylogeny. The conidial suspension (1 × 107 conidia/mL) with 0.05% Tween 80 buffer was used for pathogenicity test, by spraying 2-year-old Banana Shrub plants. Ten plants were inoculated with spore suspensions (approximately 2ml per plant). An equal number of plants were sprayed with 0.05% Tween 80 buffer to serve as a control. Fifteen days later, the inoculated plants showed similar symptoms as the original diseased plants but the controls remained asymptomatic. C. karstii was re-isolated from the infected leaves and identified by morphology and a multigene phylogeny. The pathogenicity test was repeated three times with similar results, confirming Koch's postulates. To our knowledge, this is the first report of Banana Shrub leaf blight caused by C. karstii in China. This disease reduces the ornamental and economic value of Banana Shrub, and this work will provide a basis for the prevention and treatment of the disease in the future.

Since 2020, camedor palm (Chamaedorea quezalteca) plantations in the Frailesca region, Chiapas, Mexico, have been affected by foliar diseases that induce leaf spot and anthracnose symptoms that reduce production, leaf quality and thus the loss of their commercial value. The objectives of this work were to identify the causal agents and to quantify the incidence and severity of foliar diseases in plantations of camedor palm. Two fungi were recurrently isolated, one from leaves with leaf spot symptoms and the other with anthracnose, both were identified morphologically and molecularly by sequencing the internal transcribed spacer (ITS5/ITS4), pathogenicity tests were performed on healthy plants where fungal conidia were inoculated, and in the field the incidence and severity of the disease were quantified with the support of a pictographic scale. The fungi identified were Neopestalotiopsis sp. in leaf spot symptoms and Colletotricum karstii in anthracnose, both pathogens reproduced the same symptoms observed in the field, by means of pathogenicity tests. Disease incidence and severity percentages were transformed to area under the disease progress curve (AUDPC), these diseases presented an annual average incidence and severity of 35.33% (AUDPC= 14180) and 39.16% (AUDPC=12885) for leaf spot, 19.5% (AUDPC= 7123) and 55.83% (AUDPC= 20380) for anthracnose, respectively.

Jacaranda mimosifolia D. Don is widely cultivated in southwest China (Yunnan, Sichuan, and other regions). It is widely applied in papermaking, medicine, environmental monitoring, timber, urban and rural afforestation, and soil and water conservation. In October 2020, a new brown leaf spot disease of J. mimosifolia was discovered in Xichang City (27°49' to 27°56'N, 102°16' to 102°11'E), with approximately 66.23% disease incidence. Firstly, the typical symptoms showed deep yellow necrotic lesions in the center or on the margin of the leaves. Gradually, the necrotic lesions expanded and developed into brown spots. Under humid conditions, the edges of necrotic lesions turned dark brown progressively. Finally, the leaves withered, died, and fell off. Infected tissues from ten samples were cut into small pieces of 2.5 × 2.5 mm. The surfaces of infected tissues were sterilized for 30 s in 3% sodium hypochlorite, 60 s in 75% ethanol, and rinsed three times in sterile water. They were then blot-dried with autoclaved paper towels and cultured on potato dextrose agar (PDA) at 25℃ for 3 to 8 days. After culturing for 8 days at 25℃ and 12 h/12 h light/dark on PDA, the colony diameter reached 78.2 to 82.7 mm. The colonies were light orange, turned pale pink with light orange beneath. The conidia were single-celled, aseptate, cylindrical, smooth-walled, straight, hyaline with both ends bluntly rounded, measuring 12.3 to 16.8 × 4.3 to 5.6 μm (n = 100; average=14.5 × 5.1μm). These morphological characteristics were consistent with the description of C. karstii (Zhao et al. 2021). For molecular identification, the genomic DNA of the representative isolate JM202010 was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). The internal transcribed spacer (ITS) [ITS1/ITS4 (White et al., 1990)], calmodulin (CAL) [CL1C/CL2C (Weir et al., 2012)], actin (ACT) [ACT512F/ACT-783R (Carbone & Kohn, 1999)], chitin synthase (CHS-1) [CHS-79F/CHS-345R (Carbone & Kohn, 1999)], β-tubulin (TUB2) [BT2A/BT2B (O'Donnell et al., 1997)], and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [GDF/GDR (Templeton et al. 1992)] were amplified. Sequences were deposited in GenBank (ITS: OL454787, CAL: OL518966, ACT: OL518967, CHS-1: OL518968, TUB2: OL518969, and GAPDH: OL518970). BLAST results indicated that the ITS, CAL, ACT, CHS-1, TUB2 and GAPDH sequences showed >99% identity with Colletotrichum karstii sequences at NCBI (GenBank MW494453.1, MW495036.1, MG387951.1, MW495038.1, MW495042.1, and MG602034.1). The conidial suspension (1 × 106 conidia/ml) was sprayed on the leaves of 4-year-old J. mimosifolia plants (10 plants) and inoculated for pathogenicity test. Fifteen leaves of each plant (10 pots in total) were inoculated with spore suspensions on both sides of the leaves. An equal number of control leaves was sprayed with sterilized distilled water as a control. Finally, all pots were kept in a greenhouse at 26°C under a 16 h/8 h photoperiod and 60 to 68% relative humidity. The inoculated plants showed symptoms similar to those of the original diseased plants, but the controls remained asymptomatic. Colletotrichum karstii was re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated thrice, which showed similar results, confirming Koch's postulates. To our knowledge, this is the first report of brown leaf spot on J. mimosifolia caused by C. karstii in China. C. karstii was previously reported as the causal agent of anthracnose on Fatsia japonica (Xu et al. 2020) and Nandina domestica (Li et al. 2017) in China. This finding provides an important basis for further research on the control of this disease.

Abstract In April 2019, Samanea tubulosa seedlings were observed with typical anthracnose symptoms in a forest nursery in São João Evangelista, Minas Gerais, Brazil. Based on morphological characteristics and phylogenetic analyses of ITS (5.8 S ribosomal RNA and the flanking internal transcribed spacer regions), act (actin), cal (calmodulin) and gapdh (glyceraldehyde‐3‐phosphate dehydrogenase) sequences, the fungus was identified as Colletotrichum karstii. Inoculation of the fungus in healthy S. tubulosa seedlings showed symptoms similar to those observed under natural conditions. To our knowledge, this is the first report of C. karstii causing anthracnose in S. tubulosa worldwide.