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.

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.

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.

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.

UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

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.

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.

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 β-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.

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.

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.