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BACKROUD: Keratitis caused by Lasiodiplodia theobromae is rare and typically associated with a poor prognosis. Current literature lacks sufficient evidence on effective management of patients with this condition. CASE PRESENTATION: A 74-year-old former agricultural worker presented with a red right eye, discomfort, and decreased visual acuity, progressing over three days without treatment. Examination revealed type 2 diabetes and a non-perforating, spiculated corneal abscess with a hypopyon in the right eye. Initial treatment included a triple antibiotic therapy and supportive care. Direct mycological examination identified numerous septate mycelial filaments. Antifungal treatment with natamycin and voriconazole, both topically and orally, was initiated. Cultures confirmed Lasiodiplodia theobromae. The patient showed significant improvement. Treatment continued for eight weeks, with a final visual acuity of 20/50 due to a stromal scar. CONCLUSION: An extensive literature review conducted in November 2023, using databases such as PubMed and Google Scholar with the keywords "lasiodiplodia" and "keratitis" yielded no previous cases of this specific condition being managed solely with the combined use of natamycin and voriconazole. This antifungal combination is commonly included in most management protocols for fungal keratitis. Factors such as the use of corticosteroids and delayed diagnosis were noted to adversely affect the prognosis. This case and this systematic review underscores the potential for non-surgical management options in severe fungal keratitis.

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We provide the first case report of peritoneal dialysis (PD)-associated peritonitis due to Lasiodiplodia theobromae, a known plant pathogen causing rotting and dieback in post-harvest citrus fruit, in immunocompetent patient with fungal colonization inside the PD catheter lumen. A root cause analysis suspected the patient's umbilical infection as the source of contamination. The fungal infection was established through microscopic examination of the PD catheter lumen and galactomannan testing in both serum and effluent. The species of pathogen was confirmed by DNA barcoding. The patient responded well to timely PD catheter removal and a 2-week course of oral voriconazole. Preventive strategies should prioritize hygiene practices, including umbilical care, to mitigate the risk of contamination and subsequent infections of fungal pathogens.

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Cassava root rot disease caused by the fungal pathogens Fusarium solani and Lasiodiplodia theobromae produces severe damages on cassava production. This research was conducted to produce and assess silver nanoparticles (AgNPs) synthesized by Trichoderma harzianum for reducing root rot disease. The results revealed that using the supernatants of T. harzianum on a silver nitrate solution changed it to reddish color at 48 h, indicating the formation of AgNPs. Further characterization was identified using dynamic light scattering (DLS) and scanning electron microscope (SEM). DLS supported that the Z-average size is at 39.79 nm and the mean zeta potential is at - 36.5 mV. SEM revealed the formation of monodispersed spherical shape with a diameter between 60-75 nm. The antibacterial action of AgNPs as an antifungal agent was demonstrated by an observed decrease in the size of the fungal colonies using an increasing concentration of AgNPs until the complete inhibition growth of L. theobromae and F. solani at > 58 µg mL-1 and at ≥ 50 µg mL-1, respectively. At in vitro conditions, the applied AgNPs caused a decrease in the percentage of healthy aerial hyphae of L. theobromae (32.5%) and of F. solani (70.0%) compared to control (100%). The SR-FTIR spectra showed the highest peaks in the first region (3000-2800 cm-1) associated with lipids and fatty acids located at 2962, 2927, and 2854 cm-1 in the AgNPs treated samples. The second region (1700-1450 cm-1) consisting of proteins and peptides revealed the highest peaks at 1658, 1641, and 1548 cm-1 in the AgNPs treated samples. The third region (1300-900 cm-1), which involves nucleic acid, phospholipids, polysaccharides, and carbohydrates, revealed the highest peaks at 1155, 1079, and 1027 cm-1 in the readings from the untreated samples. Finally, the observed root rot severity on cassava roots treated with AgNPs (1.75 ± 0.50) was significantly lower than the control samples (5.00 ± 0.00).

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Lasiodiplodia theobromae is a dematiaceous fungus which rarely causes keratitis and is mostly resistant to the commonly used antifungal drugs. Here, we report three cases of keratitis caused by L.theobromae from Assam. All the cases were successfully treated with 1% voriconazole and surgical debridement. To the best of our knowledge and literature search, this is the first case series of keratitis caused by L.theobromae reported from eastern India.

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Lasiodiplodia theobromae, as one of the causative agents associated with Chinese hickory trunk cankers, has caused huge economic losses to the Chinese hickory industry. Although the biological characteristics of this pathogen and the occurrence pattern of this disease have been well studied, few studies have addressed the related mechanisms due to the poor molecular and genetic study basis of this fungus. In this study, we sequenced and assembled L. theobromae strain LTTK16-3, isolated from a Chinese hickory tree (cultivar of Linan) in Linan, Zhejiang province, China. Phylogenetic analysis and comparative genomics analysis presented crucial cues in the prediction of LTTK16-3, which shared similar regulatory mechanisms of transcription, DNA replication, and DNA damage response with the other four Chinese hickory trunk canker-associated Botryosphaeria strains including, Botryosphaeria dothidea, Botryosphaeria fabicerciana, Botryosphaeria qingyuanensis, and Botryosphaeria corticis. Moreover, it contained 18 strain-specific protein clusters (not conserved in the other L. theobromae strains, AM2As and CITRA15), with potential roles in specific host-pathogen interactions during the Chinese hickory infection. Additionally, an efficient system for L. theobromae protoplast preparation and polyethylene glycol (PEG) -mediated genetic transformation was firstly established as the foundation for its future mechanisms study. Collectively, the high-quality genome data and the efficient transformation system of L. theobromae here set up the possibility of targeted molecular improvements for Chinese hickory canker control.IMPORTANCEFungi with disparate genomic features are physiologically diverse, possessing species-specific survival strategies and environmental adaptation mechanisms. The high-quality genome data and related molecular genetic studies are the basis for revealing the mechanisms behind the physiological traits that are responsible for their environmental fitness. In this study, we sequenced and assembled the LTTK16-3 strain, the genome of Lasiodiplodia theobromae first obtained from a diseased Chinese hickory tree (cultivar of Linan) in Linan, Zhejiang province, China. Further phylogenetic analysis and comparative genomics analysis provide crucial cues in the prediction of the proteins with potential roles in specific host-pathogen interactions during the Chinese hickory infection. An efficient PEG-mediated genetic transformation system of L. theobromae was established as the foundation for the future mechanisms exploration. The above genetic information and tools set up valuable clues to study L. theobromae pathogenesis and assist in Chinese hickory canker control.

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In recent years, avocado branch blight has gradually become one of the major diseases causing mortality of avocado trees, which seriously affects the economic development of avocado planting regions. In order to investigate the cause of the disease, the pathogens were isolated from the interroot of avocado trees with the onset of the disease and identified as Lasiodiplodia theobromae. At the same time, three Bacillus velezensis strains, YK194, YK201, and YK268, with better antagonistic effects and high stability against L. theobromae, were isolated from the rhizospheric soil of healthy avocado plants. The results of branch experiments and field trials showed that the avocado leaves as well as branches treated with the strains YK194, YK201, and YK268 did not develop disease, and the incidence of avocado trees was significantly reduced. In the branch experiments, the biological control effect of the strains YK194, YK201, and YK268 reached 62.07, 52.70, and 72.45%, respectively. In the field experiments, it reached 63.85, 63.43, and 73.86%, respectively, which indicated that all these three strains possessed good biological control effects on avocado branch blight. Further investigation on the mechanism of action of antagonistic strains revealed that B. velezensis YK268 could produce lipopeptides, namely, surfactin, fengycin, and iturin, which could significantly inhibit the spore germination of L. theobromae. Consequently, these three isolates have potential as biocontrol agents against L. theobromae.

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High temperatures associated with a fluctuating climate profoundly accelerate the occurrence of a myriad of plant diseases around the world. A comprehensive insight into how plants respond to pathogenic microorganisms under high-temperature stress is required for plant disease management, whereas the underlying mechanisms behind temperature-mediated plant immunity and pathogen pathogenicity are still unclear. Here, we evaluated the effect of high temperature on the development of grapevine canker disease and quantified the contribution of temperature variation to the gene transcription reprogramming of grapevine and its pathogenic agent Lasiodiplodia theobromae using a dual RNA-seq approach. The results showed that both grapevine and the pathogen displayed altered transcriptomes under different temperatures, and even the transcription of a plethora of genes from the two organisms responded in different directions and magnitudes. The transcription variability that arose due to temperature oscillation allowed us to identify a total of 26 grapevine gene modules and 17 fungal gene modules that were correlated with more than one gene module of the partner organism, which revealed an extensive web of plant-pathogen gene reprogramming during infection. More importantly, we identified a set of temperature-responsive genes that were transcriptionally orchestrated within the given gene modules. These genes are predicted to be involved in multiple cellular processes including protein folding, stress response regulation, and carbohydrate and peptide metabolisms in grapevine and porphyrin- and pteridine-containing compound metabolisms in L. theobromae, implying that in response to temperature oscillation, a complex web of signaling pathways in two organism cells is activated during infection. This study describes a co-transcription network of grapevine and L. theobromae in the context of considering temperature variation, which provides novel insights into deciphering the molecular mechanisms underlying temperature-modulated disease development.

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Purpose: We present the clinical and histopathological findings of a geographically unique Lasiodiplodia theobromae fungal keratitis case in North Carolina. L. theobromae is a rare cause of fungal keratitis, and all but one of the 51 previously reported cases have occurred in patients living in the tropics. Observations: A man in his early 50s developed L. theobromae keratitis after being struck in the left eye by a piece of debris while using a flexible-cord weed trimmer. Intracapsular lensectomy and penetrating keratoplasty were necessary when initial antimicrobial therapy was ineffective. The best-corrected visual acuity was 20/40 four years postoperatively. Conclusions and Importance: Our patient is only the second example of L. theobromae keratitis in a patient living in a sub-tropical climate and the first case in the U.S.A. outside of Florida. Additional in-vitro antibiotic sensitivity testing and documentation of more clinical cases are needed to define the optimal therapy for Lasiodiplodia theobromae keratitis.

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Stem-end rot disease has been causing damage to the production of pomelos in Vietnam. The cur-rent study aimed to (i) isolate fungal pathogens causing pomelo stem-end rot disease (PSERD) and (ii) discover Trichoderma spp. that had an antagonistic ability against pathogens under in vitro conditions. Fungi causing PSERD were isolated from pomelo fruits with symptoms of stem-end rot disease and collected from pomelo farms in Ben Tre province, Vietnam. Moreover, 50 fungal strains of Trichoderma spp. also originated from soils of these pomelo farms in Ben Tre province and were dual-tested with the fungal pathogen on the PDA medium. The results demonstrated that 11 pathogenic fungi causing PSERD were isolated from the fruit and showed mycelial growth of roughly 5.33-8.77 cm diameter at 72 h after inoculation. The two fungi that exhibited the fast-est growth, namely, S-P06 and S-P07, were selected. ITS sequencing of the S-P06 and S-P07 fungi resulted in Lasiodiplodia theobromae. All the 50 Trichoderma spp. strains were allowed to antago-nize against the S-P06 and S-P07 strains under in vitro conditions. The greatest antagonistic effi-ciency was found in Trichoderma spp. T-SP19 at 85.4-86.2% and T-SP32 at 84.7-85.4%. The two antagonists were identified as Trichoderma asperellum T-SP19 and T-SP32. The selected strains of Trichoderma asperellum were potent as a biological control for fruit plants.

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Lasiodiplodia theobromae, a grapevine trunk pathogen, is becoming a significant threat to vineyards worldwide. In Peru, it is responsible for Botryosphaeria dieback in many grapevine-growing areas and it has spread rapidly due to its high transmissibility; hence, control measures are urgent. It is known that some endophytic bacteria are strong inhibitors of phytopathogens because they produce a wide range of antimicrobial molecules. However, studies of antimicrobial features from endophytic bacteria are limited to traditional confrontation methods. In this study, a MALDI mass spectrometry-based approach was performed to identify and characterize the antifungal molecules from Bacillus velezensis M1 and Bacillus amyloliquefaciens M2 grapevine endophytic strains. Solid medium antagonism assays were performed confronting B. velezensis M1 - L. theobromae and B. amyloliquefaciens M2 - L. theobromae for antifungal lipopeptides identification. By a MALDI TOF MS it was possible identify mass spectra for fengycin, iturin and surfactin protoned isoforms. Masses spectrums for mycobacillin and mycosubtilin were also identified. Using MALDI Imaging MS we were able to visualize and relate lipopeptides mass spectra of fengycin (1463.9 m/z) and mycobacillin (1529.6 m/z) in the interaction zone during confrontations. The presence of lipopeptides-synthesis genes was confirmed by PCR. Liquid medium antagonism assays were performed for a proteomic analysis during the confrontation of B. velezensis M1 - L. theobromae. Different peptide sequences corresponding to many antifungal proteins and enzymes were identified by MALDI TOF MS/MS. Oxalate decarboxylase bacisubin and flagellin, reported as antifungal proteins, were identified at 99 % identity through peptide mapping. MALDI mass spectrometry-based identification of antifungal molecules would allow the early selection of endophytic bacteria with antifungal features. This omics tool could lead to measures for prevention of grapevine diseases and other economically important crops in Peru.

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Lasiodiplodia theobromae attacks over 500 plant species and is an important pathogen of tropical and subtropical fruit. Due to global warming and climate change, the incidence of disease associated with L. theobromae is rising. Virulence tests performed on avocado and mango branches and fruit showed a large diversity of virulence of different L. theobromae isolates. Genome sequencing was performed for two L. theobromae isolates, representing more virulent (Avo62) and less-virulent (Man7) strains, to determine the cause of their variation. Comparative genomics, including orthologous and single-nucleotide polymorphism (SNP) analyses, identified SNPs in the less-virulent strain in genes related to secreted cell wall-degrading enzymes, stress, transporters, sucrose, and proline metabolism, genes in secondary metabolic clusters, effectors, genes involved in the cell cycle, and genes belonging to transcription factors that may contribute to the virulence of L. theobromae. Moreover, carbohydrate-active enzyme analysis revealed a minor increase in gene counts of cutinases and pectinases and the absence of a few glycoside hydrolases in the less-virulent isolate. Changes in gene-copy numbers might explain the morphological differences found in the in-vitro experiments. The more virulent Avo62 grew faster on glucose, sucrose, or starch as a single carbon source. It also grew faster under stress conditions, such as osmotic stress, alkaline pH, and relatively high temperature. Furthermore, the more virulent isolate secreted more ammonia than the less-virulent one both in vitro and in vivo. These study results describe genome-based variability related to L. theobromae virulence, which might prove useful for the mitigation of postharvest stem-end rot. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

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Adenia globosa, as an excellent indoor ornamental plant, is planted in Tropical Botanical Museum, Nanjing Zhongshan Botanical Garden, Jiangsu Province, China. In September 2022, a new stem basal rot disease was observed on A. globosa seedlings, being planted here. Stem basal rot were observed on approximately 80% of A. globosa seedlings. The basal stem of cutting seedlings appeared decayed, and stem tip eventually turned dry due to water loss (Figure S1A). To isolate the pathogen, three diseased stems were collected from three cuttings planted in different pots of the Tropical Botanical Museum. The stem sections (3 to 4 mm) were excised from the margins between healthy and diseased tissues, surface sterilized in 75% ethanol for 30 s and 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃ in the dark. Pure cultures were obtained by monosporic isolation. Eight isolates were obtained, and all identified as Lasiodiplodia sp.. The colonies morphology of cultures, growing on PDA were cotton-like, the primary mycelia were black gray after 7 days, and the reverse sides of PDA plates were similar to front sides in color (Figure S1B). A representative isolate, QXM1-2 was selected for the further study. Conidia of QXM1-2 were oval or elliptic, with a mean size of 11.6 µm×6.6 µm (n=35). The conidia are colorless and transparent in the early stage, and become dark brown with one-septum in the later stage (Figure S1C). The conidiophores produced conidia after nearly four weeks of cultivation on PDA plate (Figure S1D). The conidiophore was a transparent cylindrical structure, with a size of (6.4-18.2) µm × (2.3-4.5) µm ( n = 35). These characteristics were consistent with the description of Lasiodiplodia sp. (Alves et al. 2008). The internal transcribed spacer regions (ITS), translation elongation factor 1-alpha (TEF1α) and ß-tubulin (TUB) genes (GenBank Accession No.OP905639, No.OP921005, and No.OP921006, respectively) were amplified and sequenced with the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Alves et al. 2008) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. They had 99.8-100% homology to the ITS (504/505 bp) of Lasiodiplodia theobromae strain NH-1 (MK696029), TEF1α (316/316 bp) of strain PaP-3 (MN840491), and TUB (459/459 bp) of isolate J4-1 (MN172230). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate QXM1-2 clustered in the L. theobromae clade with 100% bootstrap support (Figure S2). To test pathogenicity, three A. globosa cutting seedlings that previously had been wounded with a sterile needle were inoculated with 20 µL conidia suspension (1×106 conidia/mL) on the stem base. The seedlings inoculated with 20 µL sterile water was used as the control. All plants were covered with clear polyethylene bags to keep moisture in a greenhouse (25℃, 80% relative humidity). The experiment was repeated three times. After 7 days post-inoculation, typical stem rot were found on the treated cutting seedlings and the control seedlings did not have any symptoms (Figure S1E-F). The same fungus, identified by morphological characteristics and sequencing using ITS, TEF1α and TUB genes, was isolated from the diseased tissues of the inoculated stems to complete Koch's postulates. This pathogen has been reported infecting the branch of castor bean (Tang et al. 2021) and root of Citrus (Al-Sadi et al. 2014). For our knowledge, this is the first report of L. theobromae infecting A. globosa in China. This study provides an important reference for the biology, epidemiology of L. theobromae.

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We report an uncommon instance of rhinosinusitis by Lasiodiplodia theobromae in a known diabetic patient. A melanized fungus called Lasiodiplodia theobromae causes a typical plant disease that rots fruits and plants. Infections in humans are currently limited. Mostly from tropical and subtropical regions, there have been few reported occurrences. The fungus has been associated with clinical manifestations such as onychomycosis, corneal ulcers, and phaeohyphomycosis. Identification by phenotype was inconclusive. DNA sequencing was used for final identification. Amphotericin B and surgical debridement were effective treatments for the patient.

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The effector proteins secreted by a pathogen not only promote the virulence and infection of the pathogen but also trigger plant defense response. Lasiodiplodia theobromae secretes many effectors that modulate and hijack grape processes to colonize host cells, but the underlying mechanisms remain unclear. Herein, we report LtGAPR1, which has been proven to be a secreted protein. In our study, LtGAPR1 played a negative role in virulence. By co-immunoprecipitation, 23 kDa oxygen-evolving enhancer 2 (NbPsbQ2) was identified as a host target of LtGAPR1. The overexpression of NbPsbQ2 in Nicotiana benthamiana reduced susceptibility to L. theobromae, and the silencing of NbPsbQ2 enhanced L. theobromae infection. LtGAPR1 and NbPsbQ2 were confirmed to interact with each other. Transiently, expressed LtGAPR1 activated reactive oxygen species (ROS) production in N. benthamiana leaves. However, in NbPsbQ2-silenced leaves, ROS production was impaired. Overall, our report revealed that LtGAPR1 promotes ROS accumulation by interacting with NbPsbQ2, thereby triggering plant defenses that negatively regulate infection.

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The EFSA Plant Health Panel performed a pest categorisation of Lasiodiplodia pseudotheobromae, a clearly defined fungus of the family Botryosphaeriaceae, which was first described in 2008 as a cryptic species within the L. theobromae complex. The pathogen affects a wide range of woody perennial crops and ornamental plants causing root rot, damping-off, leaf spots, twig blight, cankers, stem-end rot, gummosis, branch dieback and pre- and post-harvest fruit rots. Lasiodiplodia pseudotheobromae is present in Africa, Asia, North and South America and Oceania and has also been reported from Spain with a restricted distribution. However, there is uncertainty on the status of the pathogen worldwide and in the EU because in the past, when molecular tools (particularly multigene phylogenetic analysis) were not available, the pathogen might have been misidentified as L. theobromae. Lasiodiplodia pseudotheobromae is not included in Commission Implementing Regulation (EU) 2019/2072 and there are no interceptions in the EU. Because of the very wide host range of the pathogen, this pest categorisation focused on those hosts for which there is robust evidence that the pathogen was formally identified by a combination of morphology, pathogenicity and multilocus sequence analysis. Plants for planting, including seeds, fresh fruits and bark and wood of host plants as well as soil and other plant-growing media are the main pathways for the further entry of the pathogen into the EU. Host availability and climate suitability factors occurring in parts of the EU are favourable for the further establishment of the pathogen. In the area of its present distribution, including Spain, the pathogen has a direct impact on cultivated hosts. multilocus measures are available to prevent the further introduction and spread of the pathogen into the EU. Lasiodiplodia pseudotheobromae satisfies the criteria that are within the remit of EFSA to assess for this species to be regarded as potential Union quarantine pest.

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Lasiodiplodia theobromae is a causal agent of Botryosphaeria dieback, which seriously threatens grapevine production worldwide. Plant pathogens secrete diverse effectors to suppress host immune responses and promote the progression of infection, but the mechanisms underlying the manipulation of host immunity by L. theobromae effectors are poorly understood. In this study, we characterized LtCre1, which encodes a L. theobromae effector that suppresses BAX-triggered cell death in Nicotiana benthamiana. RNAi-silencing and overexpression of LtCre1 in L. theobromae showed impaired and increased virulence, respectively, and ectopic expression in N. benthamiana increased susceptibility. These results suggest that LtCre1 is as an essential virulence factor for L. theobromae. Protein-protein interaction studies revealed that LtCre1 interacts with grapevine RGS1-HXK1-interacting protein 1 (VvRHIP1). Ectopic overexpression of VvRHIP1 in N. benthamiana reduced infection, suggesting that VvRHIP1 enhances plant immunity against L. theobromae. LtCre1 was found to disrupt the formation of the VvRHIP1-VvRGS1 complex and to participate in regulating the plant sugar-signaling pathway. Thus, our results suggest that L. theobromae LtCre1 targets the grapevine VvRHIP1 protein to manipulate the sugar-signaling pathway by disrupting the association of the VvRHIP1-VvRGS1 complex.

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Loquat (Rhaphiolepis biabas, heterotypic synonym: Eriobotrya japonica) is an important edible and medicinal plant that is widely cultivated on 133 thousand hectares (recorded in 2022) in China. A stem brown rot was observed on young and old trees in Mengzi city (23°23' N; 103°23' E), Yunnan Province, southwest China, during October 2014 and September 2021. Incidence ranged from 20% of trees in surrounding plantations to 50% incidence of a 160 tree orchard that was the focal point of the disease survey. Circular brown lesions occurred initially on the stems and gradually covered all the epidermis of the stem, leading to irregular dents within the bark that developed a dark brown powdery appearance (Fig.1A). Larger lesions affected vascular tissues, causing diseased trees to wither and die. Diseased tissues were surface-disinfected in a 5% sodium hypochlorite solution for 3 min, rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA), and incubated in the dark at 28°C. Twenty samples were collected for tissue isolation, and 11 isolates were single-spored on water agar. In culture, the colonies on PDA were white to dark-gray, velvet, with dense hyphae, diameter 7.64 cm after 5 days. After 18 days, spherical or subglobose pycnidia were developed and semi-buried in medium, their walls were thicker and dark-brown, which were black particles surrounded by gray-black hyphae. Conidiogenous cells were hyaline, cylindrical, holoblastic, slightly swollen at the base, with rounded apex. Conidia were initially hyaline and aseptate with elliptic or ovate shape, becoming dark brown with a single septate and developing longitudinal striations along thick walls at maturity. Conidia dimensions varied from 8.0 to 12.2 × 3.8 to 6.1µm (n=50) (Fig.1D). The morphological characteristics of eleven isolates were consistent with the description of Lasiodiplodia theobromae (Alves et al. 2008). Further confirmation was also determined by sequencing the internal transcribed spacer (ITS), ß-tubulin genes, partial translation elongation factor-1α (TEF-1α) (White et al. 1990, Carbone et al. 1999, Glass et al.1995). The isolate LSB-1 was selected for DNA sequence analysis. Based on BLASTn analysis, ITS sequences (OM617921) had 98.3% similarity with L. theobromae CBS164.96 (accession AY640255), CBS124.13(accession DQ458890), CAA006 (accession DQ458891) and CBS111530 (accession EF622074), ß-tubulin sequences (OM643838) showed 99.1% similarity with L. theobromae accessions EU673110. The TEF-1α (OM643839) had 99.0% identity with L. theobromae accession EF633054. The isolate LSB-1 clustered on the same clade with other L. theobromae. Pathogenicity testing of isolate LSB-1, LSB-2, LSB-3 was conducted by inoculating the stems of l-year-old seedlings growing in pots. The epidermis at the inoculation site, 15-20 cm below the crown, was wiped with 75% alcohol cotton ball, washed three times with sterile water, and then punctured (5mm diameter) with sterile inoculation needle. A 5mm block of each isolate cultured on PDA for seven days was attached to the inoculation site. Controls were inoculated with sterile PDA blocks. The inoculation area was covered with polyethylene cling film. All inoculated seedlings were kept in controlled greenhouse at 27°C with 80% relative humidity under natural daylight conditions, and watered weekly. Each treatment was repeated three times. Eight days after inoculation, all diseased plants showed dark brown discoloration at the point of inoculation (Fig. 1G) with the bark at the inoculation site gradually raising as the disease progressed. Thirty days after inoculation, all inoculated seedlings produced typical symptoms, whereas the control seedlings remained healthy. Fungal isolates were only recovered from symptomatic stems and were morphologically identical to L. theobromae, completing Koch's postulates. According to the relevant literature, Lasiodiplodia theobromae has a broad host range, causing numerous diseases, including canker and dieback of branch (Aguilera-Cogley et al., 2021), panicle blight (Mahadevakumar et al, 2022), root rot (Abd-El Ghani and Fatouh, 2005), fruit rot(Freire et al., 2011) in diverse geographical regions. To our knowledge, this is the first report of L. theobromae causing stem brown rot of loquat in China and provides a foundation for further study of the epidemiology and integrated management of this disease.

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Kadsura coccinea (Lem.) A. C. Smith is an evergreen liana widely cultivated in China for its economic importance in traditional medicine. Many phytochemical studies on the stems and roots of K. coccinea have shown a variety of biological activities, such as anti-hepatitis, anti-HIV, and anti-tumor (Yang et al. 2020). In July 2021, symptoms of leaf spot were observed in a plantation of K. coccinea in Longan (23°03´N, 107°54´E), Guangxi province, China. The incidence of this disease was 36%, and severity varies from approximately 20 to 40% of leaf surface coverage. Symptoms began as small brown spots that expanded into irregular to nearly flower-shaped lesions. To isolate the pathogen, leaves with spots were collected, sterilized with 75% ethanol for 15 s followed by 2% sodium hypochlorite for 120 s, rinsed three times in sterilized distilled water, cut into 5 × 5 mm pieces, and placed onto potato dextrose agar (PDA) plates. The plates were kept in an incubator at 26°C in the dark for at least 2 days. A total of 27 fungal colonies of similar morphology out of 30 pieces of infected tissues were isolated. Four representative isolates (HBB1 to HBB4) were selected to study for further characterization. Fungal colonies were initially grayish-white and then turned greenish-gray on PDA. The black pycnidium and immature conidia appeared over PDA plates after 18 days. The immature conidia were colorless and transparent, elliptical, and had a single-cell structure. After 5 days, the immature conidia gradually become black and develop into mature conidia. The mature conidia were dark brown and two-celled with longitudinal striations, 20.41-29.93 × 12.42-17.19 µm (average 26.07×14.51 µm; n = 100). For DNA-based identification, the internal transcribed spacer (ITS) region, translation elongation factor 1 alpha (EF1-α), and ß-tubulin (TUB) genes of the isolates were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Sequences were submitted to GenBank (Accession nos. MW045412 to MW045415 for ITS, MW065559 to MW065562 for EF1-α, and MW065555 to MW065558 for TUB). A phylogenetic analysis was conducted using the Maximum Likelihood method on concatenated sequences of the three genes, which showed that the four Chinese isolates from K. coccinea were clustered with reference isolates of Lasiodiplodia theobromae including the ex-neotype CBS 164.96. Pathogenicity tests were performed on young, fully expanded leaves of 2-year seedlings. A 10 µL conidial suspension (1×106 conidia/mL) was inoculated on each wound on the left-half leaf and a 10 µL sterile water was inoculated on each wound on the right-half leaf (control). Each treatment was repeated three times. Inoculated leaves were wrapped in plastic bags for 5 days and plants were maintained in a growth chamber at 27°C, 85% relative humidity. Brown leaf spots appeared 5 to 6 days after inoculation, whereas the control leaves treated with sterile water showed no symptoms. All re-isolations from spots produced colonies with the same morphological characters as L. theobromae, completing Koch's postulates. To our knowledge, this is the first report of L. theobromae causing leaf spot on K. coccinea in China and worldwide. Severe leaf disease caused by L. theobromae threatens K. coccinea production. The disease threatens K. coccinea growth, and effective control measures should be identified to reduce losses.

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Green nanotechnology, a branch of nanotechnology, makes use of extract from plants or microorganisms to synthesize nanoparticles. This approach is eco-friendlier and more cost-effective than conventional methods of nanoparticle synthesis. Silver nanoparticles have interested researchers because several studies suggest that they have a wide range of applications in the field of medicine; it is known to serve as a good antimicrobial agent. This study concentrated on the synthesis of silver nanoparticles and nanoemulsion from the extract of an endophytic fungi-Lasiodiplodia theobromae. Nanoemulsion was prepared using an essential oil-tea tree oil from Melaleuca alternifolia (commonly known as tea tree). The nanoparticles were characterized using UV-visible spectra, SEM, FESEM, EDAX, XRD, and FTIR analysis. A comparative antimicrobial study was carried out between endophytic fungal extract-derived nanoparticles (EFNP) and nanoemulsion (EFNE) against two strains of Escherichia coli, through various experimental assays including Agar well diffusion method and assays that determined the minimum inhibitory concentration, minimum bactericidal concentration, and biofilm formation. From the results obtained, it was evident that both EFNP and EFNE had antibacterial activity and that the EFNE worked better than the former. This study suggested that EFNE was a good antibiotic alternative, and further in vivo studies must be done to check the efficacy.

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Bruguiera gymnorrhiza (Linn.) Sav. is a dominant tree species of mangrove forests in tropical coastal areas of China. This species is commonly used for the greening of tidal flats and seawalls in tropical and subtropical regions (Allen, & Duke 2000). A survey that was conducted from August to September 2020, in the mangrove national nature reserve at Zhanjiang, Guangdong Province, South China. Brown leaf spot symptoms were observed on Bruguiera gymnorrhiza and disease incidence was over 10% (200 investigated trees). Symptomatic small spots initially appeared at the middle or edges of leaves, enlarged irregularly, and developed into brown necrotic spots with dying curly edges. The color of the lesion's center changed to dark brown or gray. To identify the causative agent, twenty diseased leaves were sampled for pathogen isolation. Affected foliar tissues cut into 5 × 5 mm pieces, disinfected in 75% ethanol for 2 mins, rinsed in sterile distilled water, and then air dried under a sterilized filter paper. Leaf pieces were plated onto potato dextrose agar (PDA) in Petri dishes and then incubated at 28°C in darkness for 3-5 days. Hyphal tips of fungal colonies growing from the tissue pieces were subcultured onto fresh PDA to obtain pure single hyphae cultures. The fungal colonies were initially composed of white aerial mycelia, but turned gray after 7 days. Immature conidia were hyaline, subovoid, and aseptate while mature conidia becoming dark brown, one-septate with longitudinal stripes, the length/width ratio is 19.98 to 29.50 µm (average 24.37 µm; n = 50) × 11.99 to 14.45 µm (average 13.09 µm; n = 50). On the basis of morphological features all isolates were identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl (Alves et al. 2008). For DNA-based identification, the internal transcribed spacer (ITS) region gene and fragment of elongation factor 1-alpha (EF1-α) gene of the three isolates were amplified and sequenced following the methods described in a previous study (White et al. 1990, Carbone & Kohn 1999). The obtained sequences of ITS and EF1-α were deposited in GenBank with accession numbers OK644200 and OL345571. The BLAST results showed at least 99.60% similarity with the sequences of Lasiodiplodia theobromae (ITS, MT644474.1 [99.79%]; EF1-a, MK961975.1 [99.60%]). To fulfill Koch's postulates, PDA plugs with actively growing mycelium of the isolates were inoculated on the leaves of Bruguiera gymnorrhiza plants that were wounded by using a sterilized needle or scalpel. Inoculated leaves were covered with sterilized wet cotton, and the plants were kept at 28°C and 80% relative humidity. The inoculated plants showed leaf spot symptoms that were similar to those previously observed in the field after 1-2 days, whereas control leaves remained healthy. Lasiodiplodia theobromae was consistently isolated from inoculated leaves again. Lasiodiplodia theobromae (Botryosphaeriaceae) is a plurivorous pathogen in a wide variety of hosts, mostly prevalent in tropical and subtropical climate regions. It has been previously reported to cause brown leaf spot on Broussonetia papyrifera (Luo et al. 2020), foliar diseases on Camellia oleifera (Zhu et al. 2014) and Kadsura longipedunculata (Fan et al. 2020). To our knowledge, this is the first report of Lasiodiplodia theobromae causing brown leaf spot on Bruguiera gymnorrhiza plants in China and worldwide. Our findings will help to make management strategies for control of this disease on Bruguiera gymnorrhiza.