Genomic analysis of the brassica pathogen turnip mosaic potyvirus reveals its spread along the former trade routes of the Silk Road.

Plant pathogens have agricultural impacts on a global scale and resolving the timing and route of their spread can aid crop protection and inform control strategies. However, the evolutionary and phylogeographic history of plant pathogens in Eurasia remains largely unknown because of the difficulties in sampling across such a large landmass. Here, we show that turnip mosaic potyvirus (TuMV), a significant pathogen of brassica crops, spread from west to east across Eurasia from about the 17th century CE. We used a Bayesian phylogenetic approach to analyze 579 whole genome sequences and up to 713 partial sequences of TuMV, including 122 previously unknown genome sequences from isolates that we collected over the past five decades. Our phylogeographic and molecular clock analyses showed that TuMV isolates of the Asian-Brassica/Raphanus (BR) and basal-BR groups and world-Brassica3 (B3) subgroup spread from the center of emergence to the rest of Eurasia in relation to the host plants grown in each country. The migration pathways of TuMV have retraced some of the major historical trade arteries in Eurasia, a network that formed the Silk Road, and the regional variation of the virus is partly characterized by different type patterns of recombinants. Our study presents a complex and detailed picture of the timescale and major transmission routes of an important plant pathogen.

Molecular and Biological Characteristics of a Peach Latent Mosaic Viroid PC Isolate in Peach from China: Base Mutations in Hairpin Stems and Implications for Symptomatology.

Peach latent mosaic viroid (PLMVd) infects peach trees in China and induces a conspicuous albino phenotype (peach calico, PC) that is closely associated with variants containing a 12-to-14 nucleotide hairpin insertion capped by a U-rich loop. Initially, PC disease distribution was limited to parts of Italy, and it was first detected in the field in China in 2019. To explore the molecular and biological characteristics of PLMVd PC isolates in peach in China, we conducted a comprehensive analysis of disease phenotype development and investigated the data-associated pathogenicity and in vivo dynamics of the Chinese isolate PC-A2 using slash-inoculation into GF-305 peach seedlings. Inoculated seedlings displayed PC symptoms much earlier following topping treatment, and PLMVd infectivity was further assessed using bioassay and semiquantitative RT-PCR experiments. Evolutionary analysis showed that the PC isolate and its progeny variants clustered into a single phylogroup distinct from reference PC-C40 isolates from Italy and PC-K1 and PC-K2 from South Korea. Some PC-A2 progeny variants from green leaves of PC-expressing seedlings showed unbalanced point mutations in hairpin stems compared with the PC-C40 reference sequence and constituted a new stem insertion type. The results reveal associations between the recessive phenotypes of peach albino symptoms and base variation in hairpin stem insertions relative to the PC-C40/chloroplastic heat shock protein 90 reference sequence.

Plant virology in the 21st century in China: Recent advances and future directions.

Plant viruses are a group of intracellular pathogens that persistently threaten global food security. Significant advances in plant virology have been achieved by Chinese scientists over the last 20 years, including basic research and technologies for preventing and controlling plant viral diseases. Here, we review these milestones and advances, including the identification of new crop-infecting viruses, dissection of pathogenic mechanisms of multiple viruses, examination of multilayered interactions among viruses, their host plants, and virus-transmitting arthropod vectors, and in-depth interrogation of plant-encoded resistance and susceptibility determinants. Notably, various plant virus-based vectors have also been successfully developed for gene function studies and target gene expression in plants. We also recommend future plant virology studies in China.

The coat protein of the ilarvirus prunus necrotic ringspot virus mediates long-distance movement.

The coat protein (CP) of plant viruses generally has multiple functions involving infection, replication, movement and pathogenicity. Functions of the CP of prunus necrotic ringspot virus (PNRSV), the causal agent of several threatening diseases of Prunus fruit trees, are poorly studied. Previously, we identified a novel virus in apple, apple necrotic mosaic virus (ApNMV), which is phylogenetically related to PNRSV and probably associated with apple mosaic disease in China. Full-length cDNA clones of PNRSV and ApNMV were constructed, and both are infectious in cucumber (Cucumis sativus L.), an experimental host. PNRSV exhibited higher systemic infection efficiency with more severe symptoms than ApNMV. Reassortment analysis of genomic RNA segments 1-3 found that RNA3 of PNRSV could enhance the long-distance movement of an ApNMV chimaera in cucumber, indicating the association of RNA3 of PNRSV with viral long-distance movement. Deletion mutagenesis of the PNRSV CP showed that the basic motif from amino acids 38 to 47 was crucial for the CP to maintain the systemic movement of PNRSV. Moreover, we found that arginine residues 41, 43 and 47 codetermine viral long-distance movement. The findings demonstrate that the CP of PNRSV is required for long-distance movement in cucumber, which expands the functions of ilarvirus CPs in systemic infection. For the first time, we identified involvement of Ilarvirus CP protein during long-distance movement.

The ABI5-dependent down-regulation of mitochondrial ATP synthase OSCP subunit facilitates apple necrotic mosaic virus infection.

Apple necrotic mosaic virus (ApNMV) is associated with apple mosaic disease in China. However, the mechanisms of ApNMV infection, as well as host defence against the virus, are still poorly understood. Mitochondrial ATP synthase plays a fundamental role in the regulation of plant growth and development. However, mitochondrial ATP synthase function in response to virus infection remains to be defined. In the present study, a yeast two-hybrid (Y2H) screening revealed that the apple mitochondrial ATP synthase oligomycin sensitivity-conferring protein (OSCP) subunit (MdATPO) interacts with ApNMV coat protein (CP). It was further verified that overexpression of MdATPO in Nicotiana benthamiana inhibited viral accumulation. In contrast, silencing of NbATPO facilitated viral accumulation, indicating that ATPO plays a defensive role during ApNMV infection. Further investigation demonstrated that ApNMV infection accelerated abscisic acid (ABA) accumulation, and ABA negatively regulated ATPO transcription, which was related to the ability of ABA insensitive 5 (ABI5) to bind to the ABA-responsive elements (ABREs) of the ATPO promoter. Taken together, our results indicated that transcription factor ABI5 negatively regulated ATPO transcription by directly binding to its promoter, leading to the susceptibility of apple and N. benthamiana to ApNMV infection. The current study facilitates a comprehensive understanding of the intricate responses of the host to ApNMV infection.

Systemic immune-inflammation index may predict the acute kidney injury and prognosis in patients with spontaneous cerebral hemorrhage undergoing craniotomy: a single-center retrospective study.

BACKGROUND: The systemic immune-inflammation index (SII) is an emerging prognostic marker of cancer. We aimed to explore the predictive ability of the SII on acute kidney injury (AKI) and prognosis in patients with spontaneous cerebral hemorrhage (SCH) who underwent craniotomy. METHODS: Patients with SCH who underwent craniotomy between 2014 and 2021 were enrolled in this study. The epidemiology and predictive factors for AKI after SCH were analyzed. The prognostic factors for clinical outcomes in patients with SCH and AKI were further investigated. The prognostic factors were then analyzed using a logistic regression model and a receiver operating characteristic curve. RESULTS: In total, 305 patients were enrolled in this study. Of these, 129 (42.3%) patients presented with AKI, and 176 (57.7%) patients were unremarkable. The SII (odds ratio [OR], 1.261; 95% confidence interval [CI], 1.036-1.553; P = 0.020) values and serum uric acid levels (OR, 1.004; 95% CI, 1.001-1.007; P = 0.005) were significant predictors of AKI after SCH craniotomy. The SII cutoff value was 1794.43 (area under the curve [AUC], 0.669; 95% CI, 0.608-0.730; P < 0.001; sensitivity, 65.9%; specificity, 65.1%). Of the patients with AKI, 95 and 34 achieved poor and good outcomes, respectively. SII values (OR, 2.667; 95% CI, 1.167-6.095; P = 0.020), systemic inflammation response index values (OR, 1.529; 95% CI, 1.064-2.198; P = 0.022), and Glasgow Coma Scale (GCS) scores on admission (OR, 0.593; 95% CI, 0.437-0.805; P = 0.001) were significant in the multivariate logistic regression analysis. The cutoff SII value was 2053.51 (AUC, 0.886; 95% CI, 0.827-0.946; P < 0.001; sensitivity, 78.9%; specificity, 88.2%). CONCLUSIONS: The SII may predict AKI in patients with SCH who underwent craniotomy and may also predict the short-term prognosis of these patients.

First Report of Angiopsora paspalicola Causing Leaf Rust on Paspalum conjugatum in Malaysia.

Paspalum conjugatum (family Poaceae), locally known as Buffalo grass, is a perennial weed that can be found in rice field, residential lawn, and sod farm in Malaysia (Uddin et al. 2010; Hakim et al. 2013). In September 2022, Buffalo grass with rust symptoms and signs were collected from the lawn located in Universiti Malaysia Sabah in the province of Sabah (6°01'55.6"N, 116°07'15.7"E). The incidence was 90%. Yellow uredinia were observed primarily on the abaxial surface of the leaves. As the disease progressed, leaves were covered with coalescing pustules. Microscopic examination of pustules revealed the presence of urediniospores. Urediniospores were ellipsoid to obovoid in shape, contents in yellow, 16.4-28.8 x 14.0-22.4 µm and echinulate, with a prominent tonsure on most of the spores. A fine brush was used to collect yellow urediniospores, and genomic DNA was extracted based on Khoo et al. (2022a). The primers Rust28SF/LR5 (Vilgalys and Hester 1990; Aime et al. 2018) and CO3_F1/CO3_R1 (Vialle et al. 2009) were used to amplify partial 28S ribosomal RNA (28S) and cytochrome c oxidase III (COX3) gene fragments following the protocols of Khoo et al. (2022b). The sequences were deposited in GenBank under accession numbers OQ186624- OQ186626 (985/985 bp) (28S) and OQ200381-OQ200383 (556/556 bp) (COX3). They were 100% similar to Angiopsora paspalicola 28S (MW049243) and COX3 (MW036496) sequences. Phylogenetic analysis using maximum likelihood based on the combined 28S and COX3 sequences indicated that the isolate formed a supported clade to A. paspalicola. Koch's postulates were performed with spray inoculations of urediniospores suspended in water (106 spores/ml) on leaves of three healthy Buffalo grass leaves, while water was sprayed on three additional Buffalo grass leaves which served as control. The inoculated Buffalo grass were placed in the greenhouse. Symptoms and signs similar to those of the field collection occurred after 12 days post inoculation. No symptoms occurred on controls. To our knowledge, this is the first report of A. paspalicola causing leaf rust on P. conjugatum in Malaysia. Our findings expand the geographic range of A. paspalicola in Malaysia. Albeit P. conjugatum is a host of the pathogen, but the host range of the pathogen especially in Poaceae economic crops need to be studied. Weed management could be an effective way to eliminate inoculum sources of A. paspalicola.

Microsurgical treatment of blood blister-like aneurysms: efficacy of clip-on-wrapping with autologous dura mater.

OBJECTIVE: To present a consecutive 20-year series of blood blister-like aneurysms (BBAs) to show that clip-on-wrapping with a Y-shaped autologous dura mater enables treatment of BBAs with a low complication rate and a satisfactory curative result. METHODS: A retrospective review was performed from patients with BBAs of the internal carotid artery (ICA) at the Affiliated Hospital of Qingdao University from 1999 to 2019. Diagnosis and treatment options were analyzed. Outcome was assessed using the modified Rankin scale (mRS). RESULTS: A total of 30 patients with BBAs of the ICA were included. Among these patients, 20 patients underwent microsurgical treatment (15 patients were treated by clip-on-wrapping with a Y-shaped autologous dura mater), the other 10 patients underwent endovascular treatment. All patients presented with subarachnoid hemorrhage (SAH). Four angiograms were initially negative. For all patients, intraoperative rupture occurred in five cases, but no postoperative aneurysm rupture occurred in this series. Three cases with clinical or radiologic cerebral infarctions were observed. The outcome was favorable in 26 patients. CONCLUSIONS: Clip-reinforced wrapping technique using a Y-shaped autologous dura mater may be an effective method for treating BBAs.

Small Brown Planthopper Nymph Infestation Regulates Plant Defenses by Affecting Secondary Metabolite Biosynthesis in Rice.

The small brown planthopper (SBPH, Laodelphax striatellus) is one of the most destructive insect pests in rice (Oryza sativa), which is the world's major grain crop. The dynamic changes in the rice transcriptome and metabolome in response to planthopper female adult feeding and oviposition have been reported. However, the effects of nymph feeding remain unclear. In this study, we found that pre-infestation with SBPH nymphs increased the susceptibility of rice plants to SBPH infestation. We used a combination of broadly targeted metabolomic and transcriptomic studies to investigate the rice metabolites altered by SBPH feeding. We observed that SBPH feeding induced significant changes in 92 metabolites, including 56 defense-related secondary metabolites (34 flavonoids, 17 alkaloids, and 5 phenolic acids). Notably, there were more downregulated metabolites than upregulated metabolites. Additionally, nymph feeding significantly increased the accumulation of seven phenolamines and three phenolic acids but decreased the levels of most flavonoids. In SBPH-infested groups, 29 differentially accumulated flavonoids were downregulated, and this effect was more pronounced with infestation time. The findings of this study indicate that SBPH nymph feeding suppresses flavonoid biosynthesis in rice, resulting in increased susceptibility to SBPH infestation.

The Virome of Piper nigrum: Identification, Genomic Characterization, Prevalence, and Transmission of Three New Viruses of Black Pepper in China.

Viral diseases are one of the main categories of diseases that cause substantial yield losses in black pepper. Disease symptoms in black pepper are generally complex and are often caused by both known and undescribed viruses. To identify and clarify the etiology of viral diseases in black pepper in Hainan, China, we conducted high-throughput sequencing (HTS) by targeting purified double-stranded RNA (dsRNA) and ribosomal RNA depleted total RNA (rRNA-depleted totRNA). Analysis of the data revealed the presence of one known virus, piper yellow mottle virus (PYMoV), and three newly identified viruses: black pepper virus F (BPVF) in the genus Fabavirus, black pepper virus E (BPVE) in the genus Enamovirus, and black pepper virus B (BPVB) in the genus Badnavirus. The dominant viruses in P. nigrum sampled in Hainan are PYMoV, with an incidence of 100%, followed by BPVF (84%, 133 of 158) and BPVB (66%, 105 of 158). Mechanical inoculation of sap extracts from source plants containing PYMoV, BPVF, and BPVB gave negative results on both herbaceous and woody host plants 60 days postinoculation (dpi). BPVF and PYMoV were successfully transmitted to virus-free seedlings of black pepper through bark grafting, while BPVB was experimentally undetectable up to 150 dpi. Seed transmission experiments showed that no target viruses were present in all 59 germinated seedlings. This study provides information on diagnosis, prevalence, and transmission of black-pepper-associated viruses.

First Report of Stem brown spot on 'Thai Gold' Selenicereus megalanthus in Malaysia caused by Nigrospora sphaerica.

'Thai Gold' yellow pitahaya (family Cactaceae, Selenicereus megalanthus) is a new crop being planted commercially in Malaysia. In May 2021, reddish-brown necrotic lesions were observed on the stems of approximately 60% of 'yellow pitahaya' plants in the field (~8 ha) located in the district Keningau of Sabah, Malaysia (5°20'53.1"N 116°06'23.0"E). As the disease progressed, the smaller lesions merged into larger irregularly shaped areas that formed dark brown in color. Stems with reddish-brown spot symptoms from ten plants were collected from the field and brought to the laboratory in sterilized paper bags. The symptom margin was excised into small blocks (5 x 5 x 5 mm). The blocks were surface sterilized based on Khoo et al. (2022), and placed on potato dextrose agar (PDA). The pathogens were isolated (three isolates were obtained) and cultured on potato dextrose agar (PDA) at 25°C for 5 days in the dark. The isolates developed floccose, white colony that darkened with age in PDA. Conidia (n = 30) were single celled, black, smooth, globose to subglobose, 13.9 to 18.7 µm in diameter, and borne singly on a hyaline vesicle at the tip of each conidiophore. Genomic DNA was extracted from fresh mycelia based on Khoo et al. (2021) and Khoo et al. (2022). Amplification of the internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-α (tef1-a) region and ß-tubulin (tub2) genes were performed using ITS1/ITS4 (White et al. 1990), EF1-728F/EF2 (O'Donnell et al. 1998; Carbone and Kohn, 1999) and T10/Bt2b (Glass and Donaldson, 1995; O'Donnell and Cigelnik, 1997) primer sets, respectively. The products were sent to Apical Scientific Sdn. Bhd. for purification and sequencing. BLASTn analysis of the newly generated ITS (OK448496, OM832586, OM832589) were 100% identical to Nigrospora sphaerica isolate 1SS (MN339998) (507/507 bp), tef1-a (OM223859, OM826971, OM826972) were 100% identical to Nigrospora sphaerica isolate F (MT708197) (497/497 bp) and tub2 (OL697400, OM826973, OM826974) were 100% identical to Nigrospora sphaerica isolate SN180517 (MN719407) (434/434 bp). The isolates established a supported clade to the related N. sphaerica type sequences, according to phylogenetic analysis using maximum likelihood based on the concatenated ITS, tef1-a and tub2 sequences. Morphological and molecular characterization matched the description of N. sphaerica (Kee et al. 2019). Koch's postulates were performed by spray inoculation (106 spores/ml) of isolate Keningau on the stem of three 'Thai Gold' yellow pitahaya plants in growth stage 4 (BBCH code: 419) (Kishore, 2016), while water was sprayed on three mock controls. The experiment was repeated using isolate Keningau02 and Keningau03 as inoculants. The inoculated stems on yellow pitahaya plants were covered with plastics for 48 h, and the plants were maintained in a greenhouse at room temperature 25 to 28°C with a relative humidity of 80 to 90%. All the inoculated stems developed symptoms 5 days post-inoculation, whereas no symptoms occurred on mock controls, thus fulfilling the Koch's postulates. No pathogen was isolated from the mock controls. The experiments were repeated two more times for each isolate. The reisolated fungi were identical to N. sphaerica morphologically and molecularly. Previously, N. sphaerica has been reported to cause stem brown spot disease on S. megalanthus in the Philippines (Taguiam et al. 2020). To our knowledge, this is the first report of N. sphaerica causing stem brown spot on 'Thai Gold' S. megalanthus in Malaysia. Our findings serve as a warning for the authorities and farmers that the disease threat has appeared for the Malaysian yellow pitahaya production.

First report of Macrophomina phaseolina causing leaf blight on Pometia pinnata in Malaysia.

Pometia pinnata (family Sapindaceae), locally known as 'Kasai', is a tropical hardwood and fruit tree species grown in Malaysia. The decoction of the bark is used for the treatment of fever, sores and colds, while the fruits are edible (Adema et al. 1996). In May 2021, irregular brown spots and necrotic lesions were observed on 'Kasai' with an incidence and severity of approximately 60% and 10% on 10 plants in a nursery (5°55'30.7"N 116°04'36.2"E) in Penampang, Sabah province. When the disease progressed, the spots coalesced into extended patches, blightening the leaves and, gradually, the entire foliage. Small pieces (5 x 5 mm) of infected leaves were excised from the infected margin, and then surface sterilized according to Khoo et al. (2022b), and plated on potato dextrose agar (PDA), and cultured at 25 °C. for 6 days. Colonies were dark brown in color initially whitish on the PDA. The color of fungal colony was dark as the culture aged. Semi-appressed mycelia were observed on the plates with abundant microsclerotia engrossed in the agar. Aggregation of hyphae formed black and round to oblong or irregular shaped microsclerotia. Thirty sclerotia from a representative isolate measured average 63-171 µM length x 57-128 µM wide. The morphological features matched the description of Macrophomina phaseolina (Abd Rahim et al. 2019). The fungal genomic DNA was extracted based on Khoo et al. (2022a and 2022b). PCR was performed using primer sets ITS1/ITS4 (White et al. 1990), EF1-728/EF2 (O'Donnell et al. 1998; Carbone and Kohn, 1999) and T1/T22 (O'Donnell and Cigelnik 1997) to amplify the internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-α (TEF-1α) region and partial ß-tubulin (TUB) gene. PCR products with positive amplicons were sent to Apical Scientific Sdn. Bhd. in Malaysia for sequencing. According to results (GenBank Accession No. OK465197, OM677767, ON237461), they were 100% identical with the reference sequence of Macrophomina phaseolina containing approximately 537 bp, 438 bp and 659 bp of the presented ITS, TEF-1α and TUB region (GenBank Accession No. MN629245, MN136199 and KF952208, respectively). The pathogen was identified as M. phaseolina based on its morphological and molecular data (Abd Rahim et al. 2019). To confirm the pathogenicity test, three non-wounded and healthy leaves of one-month-old 'Kasai' seedlings were inoculated with mycelium plug (1 x 1 cm) of M. phaseolina. Additional three 'Kasai' seedlings were inoculated with sterile PDA agar plug (1 x 1 cm) to serve as controls. The seedlings were monitored and incubated in a greenhouse at ambient temperature based on Iftikhar et al. (2022). After 6 days of inoculation, all infected leaves exhibited the symptoms as observed in the nursery, while the controls remained asymptomatic. The experiment was repeated twice. Re-isolation was performed from the symptomatic leaves and controls. The reisolated fungal isolates were identical to M. phaseolina morphologically and molecularly. No pathogens were isolated from the mock controls. M. phaseolina has been reported to cause leaf blight on Jasminium multiflorum in India (Mahadevakumar and Janardhana, 2016), and Crinum asiaticum and Hymenocallis littoralis in Malaysia (Abd Rahim et al. 2019). To our knowledge, this is the first report of M. phaseolina causing leaf blight on 'Kasai' in Malaysia and worldwide. Our findings serve as a warning for the authorities and farmers that the disease threat has appeared for 'Kasai' in Malaysia.

First Report of Neoscytalidium dimidiatum Causing stem canker on Selenicereus megalanthus in Malaysia.

Selenicereus megalanthus (family Cactaceae), commonly known as yellow pitahaya is a new crop being planted commercially in Malaysia. In May 2021, stem canker symptoms with sign of black pycnidia formed on the surface of canker (30- to 55-mm in diameter) were observed on the stem of 80% of 'yellow pitahaya' plants in the field (~8 ha) located in the district Keningau of Sabah, Malaysia (5°20'53.1"N 116°06'23.0"E). The infected stems became rotted when black pycnidia formed. To isolate the pathogen, the symptom margin was excised into four small blocks (5 x 5 x 5 mm), and the blocks were surface sterilized based on Khoo et al. (2022) before plating on potato dextrose agar (PDA). Plates were incubated at 25°C for 7 days in the dark. Two isolates were obtained and were named Keningau and Keningau02. Powdery white mycelia were initially observed in two plates, and then became dark grey with age. Dark pigmentation in plates was observed after a week of incubation at 25°C in the dark. Arthroconidia (n= 30) were hyaline to dark brown, circular or cylindrical with round to truncate ends, with zero to one septum, measuring 8.9 x 5.6 µm in size. Conidia (n= 30) exuded in milky white cirrhus from pycnidia were one-celled, aseptate, oblong, measuring 10.3 × 4.6 µm in size. When reached the maturity stage, conidia were brown and septate. Genomic DNA from Keningau and Keningau02 were extracted from fresh mycelia based on Khoo et al. (2021) and Khoo et al. (2022). Amplification of the internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-α (TEF1) region and ß-tubulin (TUB) genes were performed using ITS1/ITS4, EF1-728F/EF1-986R and T10/Bt2b primer sets, respectively (Carbone and Kohn, 1999; O'Donnell et al. 1997; White et al. 1990). The products were sent to Apical Scientific Sdn. Bhd. for sequencing. BLASTn analysis of the newly generated ITS (GenBank OK458559, OM649909), TEF1 (GenBank OM677768, OM677769) and TUB (GenBank OL697398, OM677766) indicated 99% identity to Neoscytalidium novaehollandiae strain CBS 122071 (GenBank MT592760). Phylogenetic analysis using maximum likelihood and Bayesian inference on the concatenated ITS-TEF1-TUB was constructed using IQ-Tree and MrBayes3.2.7. Neoscytalidium hyalinum, N. novaehollandiae and Neoscytalidium orchidacearum are reduced to synonymy with N. dimidiatum (Philips et al. 2013; Zhang et al. 2021). Although N. novaehollandiae is morphologically and phylogenetically similar to N. dimidiatum, but N. novaehollandiae produce muriform, Dichomera-like conidia that distinguish this species from other known Neoscytalidium species (Crous et al. 2006). No muriform, Dichomera-like conidia were observed in the Malaysia' isolates. The pathogen was identified as N. dimidiatum based on molecular data and morphological characterization (Serrato-Diaz and Goenaga, 2021). Pathogenicity tests were performed based on Mohd et al. (2013) by injection inoculation of 0.2 ml of conidial suspensions (1 x 106 conidia/ml) from isolate Keningau to three 30-month-old yellow pitahaya stems using a disposable needle and syringe. Distilled water was injected into three mock controls. The inoculated yellow pitahaya plants were covered with plastics for 48 h and incubated at 25°C. The pathogenicity test was also performed using isolate Keningau02. All inoculated stems developed symptoms as described after 6 days post-inoculation, whereas no symptoms occurred on controls, thus fulfilling Koch's postulates. The experiments were repeated two more times. The reisolated fungi were identical to the pathogen morphologically and molecularly. To our knowledge, this is the first report of N. dimidiatum causing stem canker on S. megalanthus in Malaysia. Our findings serve as a warning for the authorities and farmers that the disease threat has appeared in the Malaysian yellow pitahaya production.

First report of Anthracnose on 'Purple Dream' Solanum melongena in Malaysia Caused by Colletotrichum siamense.

'Purple Dream' eggplant (Solanum melongena) is widely grown for its edible fruits in Malaysia. In July 2021, anthracnose symptoms were observed on fruit with a disease severity of approximately 80% and an incidence of 10% in a field (14.6 m2) (5°56'50.9"N, 116°04'31.9"E) located in the Penampang district of Sabah province. The symptoms initially appeared as irregular light brown spots. As the disease progressed, the spots enlarged and merged into extensive lesion patches that appeared in concentric circles. The symptomatic fruit tissues (5 x 5 mm) were surface sterilized based on Khoo et al. (2022), and plated onto potato dextrose agar (PDA), and incubated at 25°C in the dark. Colonies with gray-white fluffy mycelia developed after 7 days, and the reverse of the colonies was dark brown. A representative isolate named Penampang was characterized morphologically and molecularly. The conidia were one-celled, cylindrical, blunt at the ends, hyaline, smooth, and measured 13.3 to 16.1 x 3.9 to 6.0 µm (n= 20). Appressoria ranged in size from 7.6 to 9.3 m x 5.5 to 6.6 µm (n= 20) and were spherical to irregular in shape and dark brown in colour. Genomic DNA was extracted from fresh mycelia of isolate Penampang based on Khoo et al. (2021) and Khoo et al. (2022). ITS1/ITS4, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and GDF1/GDR1 primer pairs were used to amplify the isolate's internal transcribed spacer region (ITS), and partial calmodulin (CAL), actin (ACT), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase genes (GAPDH) (Weir et al. 2012). PCR products were sequenced by Apical Scientific Sdn. Bhd. (Selangor, Malaysia). Sequences were deposited in GenBank under the accession numbers OL957466 (ITS), OL953035 (CAL), OL953032 (ACT), OL953038 (CHS-1), and OL953041 (GAPDH). They were 99% to 100% identical to the Colletotrichum ti ITS (NR_120143) (515 bp out of 519 bp), and C. siamense CAL (JX009714) (729 bp out of 731 bp), ACT (JX009518) (282 bp out of 282 bp), CHS-1 (JX009865) (299 bp out of 299 bp), and GAPDH (JX009924) (276 bp out of 277 bp) sequences. ITS sequences do not reliably resolve relationships within the C. gloeosporioides complex (Weir et al. 2012). The phylogenetic maximum likelihood analysis using the combined ITS, CAL, ACT, CHS-1, and GAPDH sequences indicated that the isolate was part of the C. siamense clade (100% bootstrap value) that also contained the type isolate ICMP 18578 of this species. Morphological and molecular characterization matched the description of C. siamense (Huang et al. 2021; Ismail et al. 2021). Koch's postulates were performed similarly as described by Chai et al. (2017) but using spray-inoculation (108 spores/ml) of three healthy 'Purple Dream' eggplant fruit with isolate Penampang. Water was sprayed on three additional fruits that served as controls. All the fruits were incubated at 25°C and less than 90% relative humidity. Symptoms similar to those observed in the field developed 5 days after inoculation. No symptoms occurred on controls. The experiment was repeated two more times. The reisolated fungi were identical to the pathogen morphologically and molecularly. To our knowledge, this is the first report of C. siamense causing anthracnose on fruit of 'Purple Dream' S. melongena in Malaysia as well as worldwide. Our findings expand the host range of C. siamense and indicate that the pathogen could potentially limit 'Purple Dream' eggplant production in Malaysia.

First Report of Epicoccum sorghinum Causing leaf spot on Platostoma palustre in Malaysia.

Platostoma palustre (family Lamiaceae), locally known as 'Black Cincau', is an herb processed as herbal drinks in Malaysia. In November 2021, brown lesions were observed on leaf samples of P. palustre with an incidence of approximately 10% in a nursery in Penampang, Sabah province (5°55'30.4"N 116°04'35.7"E). The lesions developed into larger chlorotic spots with aging of leaves. Five samples of infected leaves were collected, excised (5 × 5 mm), and then surface sterilized with 75% ethanol for 1 minute, washed with 2% sodium hypochlorite solution for 1 minute, rinsed, and air dried before inoculated onto potato dextrose agar (PDA). Inoculated plates were incubated at 25°C. Three isolates were isolated from the samples, which showed cottony aerial mycelia with light purple concentric rings appeared on the reverse side of the colony after 3 days. Pycnidia which were spheroid and measured 64.0 to 114.1 × 41.2 to 88.0 µm (n= 30). Conidia, unicellular, hyaline, oval and measured 3.8 to 4.9 × 2.0 to 2.7 µm (n= 30). Chlamydospores were observed, either unicellular or multicellular. NaOH test on oatmeal agar positive, brownish red. Further, the genomic DNA of pathogens (UMS, UMS02 and UMS03) was extracted from fresh mycelia (7-day-old) using lysis buffer. Large Sub Unit (LSU), ß-tubulin (tub) and RNA polymerase II (RPB2) gene were amplified using LR0R/LR7, T10/Bt2b and RPB2-5F2/RPB2-7cR primers (Rehner and Samuel, 1994; O'Donnell and Cigelnik, 1997; Liu et al. 1999) respectively. The sequences of isolate UMS, UMS02 and UMS03 which deposited in Genbank were OM238129, ON386254, ON386255 (LSU), OM048108, ON366806, ON366807 (tub), and ON003417, ON366804, ON366805 (RPB2). They had 99-100% homology to the LSU (1328/1328 bp) of Epicoccum sorghinum isolate Lido01 (OM501128), tub (422/425 bp) of isolate BJ-F1 (MF987525), and RPB2 (596/596 bp) of isolate HYCX2 (MK836295). Phylogenetic analysis by maximum likelihood method generated from the combined tub, LSU and RPB2 sequences indicated that the isolates formed a supported clade to the related Epicoccum sorghinum type sequences. Morphological, NaOH test and molecular characterization matched the description of E. sorghinum (Boerema et al. 2004; Li et al. 2020). Koch's postulates were performed by spray inoculation (106 conidia/mL) on the leaves of three healthy P. palustre seedlings with isolate UMS, while water was sprayed on three additional P. palustre seedlings served as controls. The plants were maintained in a greenhouse at room temperature 25 to 28°C with a relative humidity of 80 to 90%. All inoculated plants exhibited the symptoms similar to those of the nursery collection occurred after 8 days post inoculation. No symptoms occurred on controls. The experiment was repeated twice. The reisolated pathogen was morphologically identical to E. sorghinum. E. sorghinum was reported previously on Myrica rubra in China (Li et al. 2020). To our knowledge, this is the first report of E. sorghinum causing leaf spot on P. palustre in Malaysia. Our findings expand the host range of E. sorghinum in Malaysia.

First Report of Fusarium oxysporum Causing leaf spot on Basella rubra in Malaysia.

Basella rubra (family Basellaceae), locally known as 'Remayong Merah', is an edible perennial vine served as leafy greens in Malaysia. In May 2021, leaves with circular brown spots ranging from 3 to 10 mm wide with purple borders were found on B. rubra growing in Penampang (5°56'55.6"N 116°04'33.5"E), Sabah province. The disease severity was 80% with 10% disease incidence on 50 plants. As the disease developed, the lesions grew larger and they developed necrotic centers. Leaves with brown spot symptoms from five plants were collected from the field. Five leaf pieces (5 x 5 mm) were excised from lesion margins, surface sterilized based on Khoo et al. (2022b), before incubation on water agar at 25°C. When five pure cultures were obtained, the fungi were cultured on potato dextrose agar (PDA) at 25°C. After 5 days, fluffy white mycelia tinged with pink pigmentation showing on the underside of the colony were observed on PDA. Mycelia became violet in color as the culture aged. The isolates were incubated on carnation leaf agar at 25°C with a 12-hour light/dark photoperiod for 10 days. Sickle-shaped, thin-walled and delicate macroconidia (n= 30), predominantly 3 septate, ranging from 21.6 to 38.3 µm long by 2.7 to 4.2 µm wide in size were observed. Kidney-shaped, aseptate microconidia (n= 30) ranged from 6.2 to 11 µm long by 2.6 to 3.9 µm wide in size, and were formed on monophialides in false heads. Chlamydospores were detected both terminally and intercalarily, singly or in pairs, with smooth or rough walls. Genomic DNA was extracted from fresh mycelia of a representative isolate from Penampang based on Khoo et al. (2022a). The primers ITS1/ITS4 (White et al. 1990) and EF1/EF2 (O'Donnell et al. 1998) were used to amplify the internal transcribed spacer (ITS) rDNA and translation elongation factor 1-α (TEF1α) region, respectively based on PCR conditions as described previously (Khoo et al. 2022b). The products were sent to Apical Scientific Sdn. Bhd. for sequencing. In BLASTn analysis, ITS sequence (OK469301) was 99% (506/507 bp) identical to isolate TSE07 (MT481761) of Fusarium oxysporum, and the TEF1α sequence (OM743433) was 100% (705/705 bp) identical to isolate BLBL5 of Fusarium oxysporum. The TEF1α sequence of Penampang was analyzed at the Fusarium MLST site (https://fusarium.mycobank.org/), and had 98% similarity to TEF1α of F. oxysporum (NRRL 22551). The pathogen was identified as F. oxysporum based on morphological (Leslie and Summerell 2006) and molecular data. A volume of 0.16 ml of spore suspensions (1 × 106 conidia/ml) were inoculated on a spot on each leaf of every three healthy B. rubra seedlings at the two-leaf stage. An additional three B. rubra seedlings were mock inoculated by pipetting sterile distilled water on similar aged leaf. The seedlings were maintained in a greenhouse at 25°C with a relative humidity of 80 to 90%. Six days after inoculation, all inoculated leaves exhibited the same symptoms as observed in the field, while the controls showed no symptoms. The experiment was repeated two more times. The reisolated fungi had the same morphology and DNA sequences as the original isolate obtained from the field samples, completing Koch's postulates. F. oxysporum has been reported previously in Bangladesh and India causing leaf spot disease on B. rubra (Dhar et al. 2015; Shova et al. 2020). To our knowledge, this is the first report of F. oxysporum causing leaf spot on B. rubra in Malaysia. The identification of leaf spot caused by F. oxysporum will enable plant health authorities and farmers to identify practices to minimize disease on this important crop.

First Report of Epicoccum sorghinum Causing leaf spot on Bothriochloa ischaemum in Malaysia.

Bothriochloa ischaemum (family Poaceae) is a perennial weed that can be found in borders of agricultural fields, pastures and roadsides in Malaysia. B. ischaemum is an important phytoremediation species in copper tailings dams (Jia et al. 2020). In December 2021, chlorotic spots with brown halos were observed on leaf samples of B. ischaemum with an incidence of approximately 80% in Penampang, Sabah province (5°56'50.4"N, 116°04'32.8"E). On older leaves, the spots coalesced into larger chlorotic spots. Small pieces (5 x 5 mm) of infected leaves collected from three plants were excised, and then surface sterilized according to Khoo et al. (2022). The fungus was isolated (one isolate was obtained) and cultured on potato dextrose agar (PDA) at 25°C. After 3 days, the colony had cottony aerial mycelia with light purple concentric rings appearing on the underside of the colony. Chlamydospores were produced, either unicellular or multicellular. Conidia were unicellular, hyaline, oval, and were 3.7 to 5.1 x 1.8 to 2.6 µm (n=20). Pycnidia were spheroid, and were 66.4 to 115.3 x 43.1 to 87.4 µm (n=20). Genomic DNA was extracted from fresh mycelia of the fungus based on the extraction method described by Khoo et al. (2022). Amplification of the internal transcribed spacer (ITS) region and large subunit (LSU) of rDNA, and actin (ACT), tubulin (TUB) and RNA polymerase II second largest subunit (RPB2) genes was performed using ITS1/ITS4, LR0R/LR7, ACT512F/ACT783R, T10/Bt2b and RPB2-5F2/RPB2-7cR primers, respectively (O'Donnell and Cigelnik, 1997; Liu et al. 1999; Sung et al. 2007; Chen et al. 2021). The PCR products were sequenced at Apical Scientific Sdn. Bhd.. Sequences were deposited in GenBank as OM453926 (ITS), OM453925 (LSU), OM451236 (ACT), OM451237 (TUB) and OM863567 (RPB2). Sequences of our isolate had 100% homology to ITS of isolate UMS (OK626271) (507/507 bp), LSU of isolate UMS (OM238129) (1328/1328 bp), ACT of isolate CZ01 (MN956831) (275/275 bp), TUB of isolate BJ-F1 (MF987525) (556/556 bp) and RPB2 of isolate HYCX2 (MK836295) (596/596 bp) sequences. Phylogenetic analysis was performed using the maximum likelihood method based on the general time reversible model with a gamma distribution and invariant sites (GTR + G + I) generated from the combined ITS, TUB, LSU and RPB2 sequences, indicating that the isolates formed a supported clade to the related Epicoccum sorghinum type sequences. Morphological and molecular characterization matched the description of E. sorghinum (Li et al. 2020). Koch's postulates were performed by spray inoculation (106 spores/ml) on the leaves of three healthy B. ischaemum plants, using isolate BPL01, while sterilized water was sprayed on three additional B. ischaemum which served as the control. Symptoms similar to those occurred after 6 days post inoculation. No symptoms occurred on controls. The experiment was repeated two more times. The reisolated pathogen was morphologically and genetically identical to E. sorghinum. E. sorghinum was reported previously on Brassica parachinensis (Yu et al. 2019), Camellia sinensis (Bao et al. 2019), Myrica rubra (Li et al. 2020), Oryza sativa (Liu et al. 2020) and Zea mays (Chen et al. 2021) in China. To our knowledge, this is the first report of E. sorghinum causing leaf spot on B. ischaemum in Malaysia. Our findings expand the geographic range and host range of E. sorghinum in Malaysia. B. ischaemum which is a weed in agricultural fields is a host of the pathogen and therefore could be a potential threat to Brassica parachinensis, Camellia sinensis, Oryza sativa and Zea mays in Malaysia. Weed management could be an effective way to eliminate inoculum sources of E. sorghinum.

First report of Epicoccum sorghinum causing leaf spot on Basella alba in Malaysia.

Basella alba (family Basellaceae) is a perennial vine that serves as an edible leaf vegetable in Malaysia. In May 2021, red spots were observed on leaf samples of B. alba in Lido, Sabah Province (5°56'39.1"N, 116°04'47.6"E). The disease severity was about 20% with 10% incidence. The spots enlarged and coalesced into larger necrotic spots. Small pieces (5 x 5 mm) of infected leaves were excised from three plants, and then surface disinfected based on Khoo et al. (2022). One fungal isolate (Lido01) was isolated and cultured on potato dextrose agar (PDA) at 25°C. A single isolate with cottony aerial mycelia and pink concentric rings was observed on the upper surface of the culture. Unicellular and multicellular chlamydospores were observed, and measured 7.1 to 14.3. × 17.8 to 74.5 µm. Conidia were unicellular, hyaline, oval, and measured 3.8 to 5.2 x 1.7 to 2.7 µm (n= 20). Pycnidia were spheroid, and measured 66.2 to 114.3 x 44.1 to 86.1 µm (n= 20). Genomic DNA was extracted from fresh mycelia according to the extraction method of Khoo et al. (2022a and 2022b). ITS1/ITS4, LR0R/LR7, ACT512F/ACT783R, and T10/Bt2b primers were used to amplify the internal transcribed spacer (ITS), large subunit (LSU), actin (ACT), and tubulin (TUB) genes, respectively (O'Donnell and Cigelnik, 1997; Chen et al. 2021). PCR products were Sanger sequenced by Apical Scientific Sdn. Bhd. (Serdang, Malaysia). Sequences of isolate Lido01 were deposited in GenBank as OM501130 (ITS), OM501128 (LSU), OM513916 (ACT) and OM513917 (TUB). Respective gene sequences of this isolate showed 100% homology to ITS sequence of isolate BPL01 (OM453926) (507/507 bp), LSU sequence of isolate BPL01 (OM453925) (1328/1328 bp), ACT sequence of isolate CZ01 (MN956831) (275/275 bp) and TUB sequence of isolate BJ-F1 (MF987525) (556/556 bp). The sequences of Lido01 established a supported clade (99% bootstrap value) to the related Epicoccum sorghinum type sequences, according to phylogenetic analysis using maximum likelihood based on the concatenated ITS, ACT, and TUB sequences. Morphological characters also matched the description of E. sorghinum (Li et al. 2020). Koch's postulates were tested as described by Chai et al. (2017) with modification by spray inoculation (106 spores/ml) on the leaves of three healthy one-month-old B. alba, while sterilized distilled water served as the control treatment. Monitoring and incubation were performed in a greenhouse based on Iftikhar et al. (2022). All inoculated leaves developed symptoms as described above by 8 days post-inoculation, whereas no symptoms occurred on controls, thus fulfilling Koch's postulates. The experiment was repeated twice. The reisolated pathogen was morphologically and genetically identical to E. sorghinum. E. sorghinum was reported causing leaf spot on Brassica parachinensis (Yu et al. 2019), Camellia sinensis (Bao et al. 2019), Myrica rubra (Li et al. 2020), Oryza sativa (Liu et al. 2020) and Zea mays (Chen et al. 2021). To our knowledge, this is the first report of E. sorghinum causing leaf spot on B. alba in Malaysia. Our findings have expanded the geographic range and host range of E. sorghinum in Malaysia, though the host range of this isolate is not known.

First Report of Leaf Spot on Basella rubra Caused by Fusarium proliferatum in Malaysia.

Basella rubra (family Basellaceae), locally known as 'Remayong Merah', is the edible perennial vine served as leafy vegetable in Malaysia. In May 2021, B. rubra's leaves with circular to subcircular purple spots (ranging from 1-10 mm wide) were collected in Lido (5°56'44.6"N 116°04'46.5"E), Sabah province. The disease severity was about 60% with 20% disease incidence on fifty plants. As disease developed, the spots grew larger and necrosis were formed within the purple spots. Small pieces (5 x 5 mm) of five diseased spots were excised, and then surface sterilized based on Khoo et al. (2022b) before plating on water agar at 25°C. Once obtained the pure cultures from all diseased spots, they were incubated on potato dextrose agar at 25°C. After 7 days, white aerial mycelium with light violet pigmentation on lower side were observed on PDA. Then, the fungi were cultured on Carnation leaf agar (CLA) at 25°C and 12-h light/dark photoperiod for 10 days. Thin-walled slender and slightly curved macroconidia (n= 20) with 3 to 5 septa were ranged from 2.3 to 2.6 µm wide by 26.8 to 44.5 µm long in size. Oval microconidia (n= 20) with no septa were 2 to 2.2 µm wide by 9.5 to 15 µm long in size. Chlamydospores were absent. Monophialids with false head were observed. Isolate Lido and Lido02 were kept in the Laboratory of Genetics, Faculty of Science and Natural Resources, Universiti Malaysia Sabah for public request. Their genomic DNA were extracted from fresh mycelia of isolates based on Khoo et al. (2022a). EF1/EF2, RPB1-Fa/RPB1-G2R and RPB2-5f2/RPB2-7cr (Jiang et al. 2021) were used to amplify the translation elongation factor 1-α (TEF1) region, RNA polymerase largest subunit gene (RPB1) and RNA polymerase second largest subunit gene (RPB2) based on PCR condition in Khoo et al. (2022b). The isolate's sequences were deposited in GenBank as OM048109, OM634654 (TEF1), OM634655, OM634657 (RPB1) and OM634656, OM634658 (RPB2). They were 99 to 100% homology to TEF1 of isolate DPCT0102-2 (LC581453) (657/657 bp), RPB1 of strain ZJ05 (MT560605) (1558/1558 bp) and RPB2 of isolate GR_FP248 (MT305154) (1867/1869 bp) sequences. These sequences were polyphasic identified at the Fusarium MLST (https://fusarium.mycobank.org/), and were more than 99% similarity to Gibberella fujikuroi species complex (NRRL 25200). Gibberella fujikuroi and Fusarium fujikuroi are synonymous with Fusarium proliferatum (Leslie and Summerell 2006). The pathogen was identified as F. proliferatum based on morphological characterization, molecular data and phylogenetic analysis. Two non-wounded leaves of three one-month-old B. rubra seedlings were inoculated with mycelium plug (10 x 10 mm). Additional three B. rubra seedlings received sterile PDA agar plug (10 x 10 mm) to serve as controls. They were incubated in a glasshouse at room temperature 25°C with a relative humidity of 80 to 90%. After 8 days of inoculation, all inoculated leaves exhibited the symptoms as observed in the field, while the controls showed no symptoms, thus confirming the Koch's postulates. The experiment was repeated two more times. The reisolated pathogens were identified as F. proliferatum via PDA macroscopically, CLA microscopically and PCR amplification. F. proliferatum was reported previously causing leaf spot disease on Cymbidium orchids (Wang et al. 2018), tobacco (Li et al. 2017) and tomato (Gao et al. 2017). To our knowledge, this is the first report of F. proliferatum causing leaf spot on B. rubra in Malaysia. Infections of leaves reduce plant vigor and marketability. The identification of leaf spot caused by F. proliferatun will enable plant health authorities and farmers to identify practices to minimize disease on this important crop.

First Report of Lasiodiplodia theobromae Causing leaf spot on Aloe vera L. var. chinensis (Haw.) Berg. in Malaysia.

Aloe vera L. var. chinensis (Haw.) Berg. (family Asphodelaceae), locally known as 'Lidah Buaya', is an economically important plant as the gel from the leaves possesses anti-inflammatory, anti-arthritic, antibacterial, and hypoglycemic properties and is used for cosmetic, pharmaceutical and healing purpose in Malaysia. In July 2021, irregular black sunken spots (3- to 10-mm in diameter) were observed on the leaves of 'Lidah Buaya' plants under leaf development stage in the field located in the district Penampang of Sabah province (N5°56'37.1" E116°04'21.5"). The disease severity was about 30% with 10% incidence. The tissues surrounding the black spots became brown and dry when the plants grew older. No gel contained in the sunken zones. Symptomatic leaf tissues (5 x 5 mm) were cut from the infected margin, surface sterilised with 75% ethanol for 1 minute, washed with 2% sodium hypochlorite solution for 1 minute, rinsed, and air dried before plating on five potato dextrose agar (PDA) plates (pH 7). Plates were incubated at 25°C for 3 days in the dark. Greyish-white fluffy mycelia were observed, and then became dark grey with age. Dark pigmentation in each plate was produced after a week of incubation at 25°C. A representative isolate Penampang was further characterized morphologically and molecularly. Immature conidia were single-celled, aseptate, ellipsoid and hyaline, measuring 19.4 × 24.5 µm (n = 30). Mature conidia were brown, thick-walled and one-septate with longitudinal striations, 22.5 × 28.3 µm (n = 30). Genomic DNA was extracted from fresh mycelia of isolate Penampang based on the extraction method described by Khoo et al. (2021) with additional of mechanical disruption using micro pestle before heating. KOD One PCR master mix (Toyobo, Japan) containing hot-start modified KOD DNA polymerase was used for PCR amplification. The PCR condition were 94°C for 10 s, 55°C for 5 s and 72°C for 2 s, for 30 cycles, and initial denaturation of 94°C for 3 min and a final extension step of 72°C for 5 min. The internal transcribed spacer (ITS) region of rDNA and tubulin (TUB) genes were amplified using ITS1/ITS4 and T10/Bt2b primer sets, respectively (O'Donnell et al. 1997; White et al. 1990). The products were then sent to Apical Scientific Sdn. Bhd. for sequencing. The generated ITS (OK209451) and TUB (OL660667) were 100% identical to L. theobromae isolate MRR-161 and CPC:27690 (GenBank MW282884 and MT592639, respectively) in BLASTn analysis. Phylogenetic analysis using maximum likelihood based on the combined ITS and TUB sequences indicated that the isolates formed a supported clade (91% bootstrap value) to the related L. theobromae. The morphological and molecular characterization of the fungus matched L. theobromae described by Pecenka et al. (2021). Mycelial agar plugs (5-mm-diameter) from 7-day-old PDA culture of Penampang isolate were placed onto pinpricked leaves of three 2-month-old 'Lidah Buaya' plants. Pinpricked leaves of three 2-month-old 'Lidah Buaya' plants received sterile 5-mm-diameter PDA agar plugs to serve as controls. The inoculated 'Lidah Buaya' plants were covered with plastics for 48 h, and were incubated at 25°C. All inoculated leaves developed symptoms as described above 6 to 7 days post-inoculation, whereas no symptoms occurred on controls, thus fulfilling Koch's postulates. The experiments were repeated twice. The reisolated fungus was identical to representative isolate Penampang morphologically and molecularly. L. theobromae was reported previously on A. vera in Cuba (Urtiaga 1986) and India (Mathur 1979). To our knowledge, this is the first report of L. theobromae causing leaf spot on A. vera in Malaysia. The occurrence of this disease emphasizes the importance of disease surveillance in the region. Plant disease management strategies need to be established to reduce the losses.