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A novel disease affecting small immature fruits has surfaced in 'Saiwaihong' apples ( Malus pumila ), a recently developed variety extensively cultivated across more than 20,000 ha in China. In an effort to pinpoint the causal agent(s) responsible for this ailment, RNA sequencing analysis was conducted on four symptomatic and four asymptomatic apple samples. The results revealed a diverse range of viruses and viroids, indicating mixed viral infection in diseased samples. However, a more focused examination involving 152 symptomatic and 122 asymptomatic fruit samples, using RT-PCR and dot-blotting hybridization techniques, highlighted a close association between the disease and the presence of apple scar skin viroid (ASSVd). Among the ASSVd variants obtained from diseased 'Saiwaihong' apples, 20 were identified, and they were either identical or closely related to isolates from various apple varieties cultivated in different regions and countries. This suggests that ASSVd isolates in 'Saiwaihong' might have been introduced from other apple varieties. Furthermore, the analysis indicates the possibility of two separate introductions, as the ASSVd 'Saiwaihong' isolates exhibited two distinct phylogenetic groups. These insights provide valuable guidance for disease control strategies and emphasize the significance of ongoing monitoring for ASSVd, both in its familiar forms and potential new variants., Competing Interests: The author(s) declare no conflict of interest.

Codiaeum variegatum is a valuable ornamental plant with distinct bright yellowing and golden spots on dark green leaves, which resemble virus symptoms. To investigate the factors, especially viral agents, associated with the variegated leaf color of Codiaeum variegatum, we performed virome profiling of a single C. variegatum 'Gold Dust' leaf sample collected from Hainan, China using ribosomal RNA-depleted total RNA sequencing on an Illumina NovaSeq 6000 platform. Two novel viruses, with two variants each, belonging to the family Closteroviridae were detected and characterized: Croton golden spot-associated virus C variants 1 and 2 (CGSaVC-v1, and CGSaVC-v2) of the genus Crinivirus and Croton golden spot-associated virus A variants 1 and 2 (CGSaVA-v1 and CGSaVA-v2) of the genus Ampelovirus. Transmission electron microscopy showed long, flexuous, filamentous virus particles approximately 15 nm in diameter and 760-770 nm in length. Molecular screening of ninety-seven variegated individual plant leaves showed a high prevalence of CGSaVA-v1 (90.7%), CGSaVA-v2 (75.3 %), CGSaVC-v1 (70.1%), and CGSaVC-v2 (47.4%), while asymptomatic leaves near the meristem tip were mostly free of the target viruses. To our knowledge, this is the first study to demonstrate the significant association between closterovirids and the golden spots. The findings provide novel insights into the genetic diversity of the family Closteroviridae and inform future germplasm conservation and new cultivar development of Codiaeum Variegatum.

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., Competing Interests: The author(s) declare no conflict of interest.

Cinnamomum camphora (Lauraceae), commonly known as camphor tree, is widely grown as an ornamental and is used as a source of camphor in Malaysia. In June 2021, leaves of three camphor trees with anthracnose symptoms were collected from a park (6°02'00.8"N, 116°07'18.5"E) at the Universiti Malaysia Sabah in Sabah province. The average disease severity across diseased plants was about 60% with 30% incidence on 10 surveyed plants. The disease severity on disease area of 10 leaves from each three diseased plants was estimated using ImageJ software. The disease incidence was determined based on Sharma et al. (2017). Gray spots were observed primarily on the surface of the leaves. After a week, the spots coalesced into larger patches, and anthracnose developed. Small pieces (5 x 5 mm) of symptomatic leaf tissue from three camphor trees were excised from the margin between healthy and symptomatic tissue. The pieces were surface-sterilized with 75% ethanol for 1 minute, washed with 2% sodium hypochlorite solution for 1 minute, rinsed, and air dried before plating in three Petri dishes with Potato dextrose agar, and incubated for 7 days at 25°C in the dark. After 7 days, all the PDA plates had abundant gray-white fluffy hyphae. Mycelium was dark brown when observed from the underside of the plate. The isolates UMS02, UMS04 and UMS05 were characterized morphologically and molecularly. The conidia were one-celled, cylindrical, hyaline, and smooth, with blunt ends, and ranged in size from 13.9 to 16.3 x 3.8 to 6.1 μm ( n = 20). Appressoria were round to irregular in shape and dark brown in color, with size ranging from 7.8 to 9.8 μm x 5.3 to 6.8 μm ( n = 20). Genomic DNA was extracted from fresh mycelium of the isolates based on Khoo et al. (2022a). Amplification of the internal transcribed spacer (ITS) region, calmodulin ( CAL ), actin ( ACT ), chitin synthase ( CHS-1 ), and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) genes of the isolate was performed using primer pairs ITS1/ITS4, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and GDF1/GDR1 (Weir et al. 2012). PCR products with positive amplicons were sent to Apical Scientific Sdn. Bhd. for sequencing. Sequences of the isolates were deposited in GenBank as OK448747, OM501094, OM501095 (ITS), OL953034, OM513908, OM513909 ( CAL ), OL953031, OM513910, OM513911 ( ACT ), OL953037, OM513912, OM513913 ( CHS-1 ), and OL953040, OM513914, OM513915 ( GAPDH ). They were 100% identical to ITS (MN296082), CAL (MN525840), ACT (MW341257, MN525819), CHS-1 (MT210318), and GAPDH (MT682399, MN525882) sequences of Colletotrichum siamense . Phylogenetic analysis using maximum likelihood on the concatenated ITS, CAL , ACT , CHS-1 and GAPDH sequences indicated that the isolates formed a clade (82% bootstrap support) to C. siamense . Morphological and molecular characterization matched the description of C. siamense (Huang et al. 2022). Koch's postulates were performed by spraying a spore suspension (10 6 spores/ml) on leaves of three healthy two-month-old camphor trees, while water was sprayed on three additional camphor trees which served as control. The inoculated camphor trees were covered with plastics for 48 h at 25°C in the dark, and then placed in the greenhouse. Monitoring and incubation were performed based on Chai et al. (2017) and Iftikhar et al. (2022). Symptoms similar to those observed in the field occurred 8 days post-inoculation. No symptoms occurred on controls. The experiment was repeated two more times. C. siamense has been reported causing anthracnose on camphor tree in China (Liu et al. 2022), Citrus spp. in Mexico (Pérez-Mora et al. 2021), and Crinum asiaticum and eggplant in Malaysia (Khoo et al. 2022b, 2022c). To our knowledge, this is the first report of C. siamense causing anthracnose on C. camphora in Malaysia. Our findings expand the geographic range of C. siamense and indicate it could be a potential threat limiting the camphor production of C. camphora in Malaysia.

Crinum asiaticum (family Amaryllidaceae), locally known as 'Pokok Bakung', is an ornamental medicinal plant grown in Malaysia. It contains chemical compounds used for antimicrobial, antioxidant, antitumor, antiemetic and wound healing (Patel, 2017). In July 2021, 'Pokok Bakung' leaves with anthracnose symptoms were collected from a park of Universiti Malaysia Sabah in the Sabah province. The disease severity was about 100% with 20% incidence. Red spots were primarily found on the leaf surfaces. Anthracnose developed as the disease progressed, and acervuli were observed in the spots. Small pieces of infected leaves (5 x 5 mm) were excised from spot margins, surface sterilized based on Khoo et al. (2022a), placed on potato dextrose agar (PDA) in Petri dishes, which were incubated for 5 days at 25°C in the dark. The colonies formed on the PDA plates were abundant with gray-white fluffy mycelia after 5 days, and the reverse view revealed brown. UMS01, a representative isolate, was used to morphologically and molecularly characterize the fungus. Conidia were one-celled, cylindrical, hyaline, smooth, and blunt at the ends, measuring 13.8 to 16.5 x 3.6 to 6.7 µm (n = 20). Appressoria ranged in size from 7.6 to 9.3 x 5.5 to 6.9 µm (n= 20) and were ovoid to clavate, spherical to irregular in shape and dark brown in color. Genomic DNA was extracted from fresh mycelia of isolate UMS01 based on Khoo et al. (2021) with the addition of mechanical disruption using a micro pestle before heating at 95°C. PCR amplification was performed based on Khoo et al. (2022a) using ITS1/ITS4, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and GDF1/GDR1 primer pairs to amplify the internal transcribed spacer (ITS) region, calmodulin ( CAL ), actin ( ACT ), chitin synthase ( CHS-1 ), and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) (Weir et al. 2012). PCR products with positive amplicons were sent to Apical Scientific Sdn. Bhd. for sequencing. The sequences were deposited in GenBank under accession numbers OK458683 (ITS), OL953033 ( CAL ), OL953030 ( ACT ), OL953036 ( CHS-1 ), and OL953039 ( GAPDH ). Before BLAST, the search set were adjusted to exclude model sequences (XM/XP) and the uncultured/environmental sample sequences, and limit to sequences from type material. They were 99-100% similar to the Colletotrichum siamense ITS (JX010171), CAL (JX009714), ACT (FJ907423) and CHS-1 (JX009865), and Colletotrichum changpingense GAPDH (MZ664048) type sequences. The GAPDH marker did not reliably resolve the relationships within the C. gloeosporioides complex (Vieira et al. 2020). Phylogenetic analysis using maximum likelihood based on the combined ITS, CAL , ACT , CHS-1 and GAPDH indicated that the isolate formed a supported clade (100% bootstrap value) to the most related C. siamense . Morphological and molecular characterization matched the description of C. siamense (Huang et al. 2021). Pathogenicity tests were performed to fulfil Koch's postulates by spraying a spore suspension (10 6 spores/ml) on the leaves of three healthy four-month-old 'Pokok Bakung' plants, while three additional plants were sprayed with water as a control. The inoculated plants were covered with plastics for 48 h at 25°C in the dark. Incubation was performed based on Iftikhar et al. (2022). Symptoms similar to those of the field collection occurred after 6 days post inoculation. No symptoms occurred on the control plants. The experiment was repeated two more times. The reisolated fungal isolates were identical to C. siamense morphologically and molecularly. Previously, C. siamense has been reported to cause anthracnose on Allamanda cathartica (Huang et al. 2021) and avocado (Li et al. 2022) in China, and 'Purple Dream' eggplant in Malaysia (Khoo et al. 2022b). Colletotrichum fructicola has been reported to cause anthracnose on C. asiaticum in China (Qing et al. 2020). To our knowledge, this is the first report of C. siamense causing anthracnose on C. asiaticum in Malaysia. Our findings expand the geographic range of C. siamense and indicate that it could be a potential threat limiting the growth and production of C. asiaticum in Malaysia.

Ixora chinensis (family Rubiaceae), locally known as 'Bunga Jejarum', is widely grown as an ornamental shrub and as sources for phytochemicals with medicinal properties in Malaysia. In May 2021, irregular brown spots were found on the leaves of some 'Bunga Jejarum' in Universiti Malaysia Sabah (6°02'01.0"N 116°07'20.2"E) located in Sabah province. As the disease progressed, the spots enlarged and coalesced into large necrotic areas giving rise to drying of infected leaves. The disease severity was about 70% with 20% incidence. Five symptomatic leaves (5 x 5 mm) from five plants were excised and sterilized based on Khoo et al. (2022) before plated on five potato dextrose agar (PDA) and cultured at 25°C. After 5 days, white to pale honey and dense mycelia with lobate edge were observed on all PDA plates. Globose, black conidiomata semi-immersed on PDA were observed after a week. Two to four hyaline filamentous appendages 7.7 to 17.6 μm long attached to fusoid conidia (11.8 to 20.9 x 5.7 to 7.6 μm, n = 20), which consisted of a hyaline apical cell, basal cell, and three versicolored median cells. The upper two median cells were dark brown, while the lowest median cell was pale brown. The isolate of the causal pathogen was characterized molecularly. Genomic DNA of isolate UMS01 was extracted based on Khoo et al. (2021) and Khoo et al. (2022). Amplification of the internal transcribed spacer ( ITS ), tubulin ( TUB ) and translation elongation factor 1-α ( TEF ) region was performed based on Khoo et al. (2022) using primers ITS1/ITS4 (White et al. 1990), T1/Bt2b (Glass and Donaldson, 1995; O'Donnell and Cigelnik, 1997) and EF1-728/EF2 (O'Donnell et al. 1998; Carbone and Kohn, 1999), respectively. PCR products with positive amplicons were sent to Apical Scientific Sdn. Bhd. for sequencing. The isolate's sequences were deposited in GenBank as OM320626 ( ITS ), OM339539 ( TUB ) and OM339540 ( TEF ). They were 99% to 100% identical to ITS (KM199347) (545 out of 545 bp), TUB (KM199438) (768 out of 769 bp) and TEF (KM199521) (480 out of 481 bp) of the type sequences (CBS 600.96). Phylogenetic analysis using the maximum likelihood method based on the combined ITS , TEF and TUB sequences placed the isolate UMS01 in the same clade as the isolate CBS 600.96 of Neopestalotiopsis cubana . Thus, the pathogen was identified as N. cubana based on the morphological description from Pornsuriya et al. (2020), molecular data in Genbank database and multigene sequence analysis. To further confirm its pathogenicity, the first and second leaves of three 'Bunga Jejarum' plants were inoculated by pipetting 1 ml aliquots of a 1 × 10 6 conidia/ml spore suspension. Three additional 'Bunga Jejarum' plants were mock inoculated by pipetting 1 ml of sterile distilled water on similar age leaves. The plants were covered with plastic bags after inoculation for 48 h before placing them in a glasshouse under room temperature. The leaves were sprayed with water to keep the leaf surfaces moist along the experiment. The incubation and disease observation were performed based on Chai et al. (2017) and Iftikhar et al. (2022). After 7 days post-inoculation, all infected leaves exhibited the symptoms observed in the field, whereas the controls showed no symptoms. The same fungus was isolated from the diseased leaves and, thus confirmed Koch's postulates. The experiment was repeated two more times. The reisolated fungi were visually and genetically identical to the original isolate obtained from the field samples. To our knowledge, this is the first report of N. cubana causing leaf blight on 'Bunga Jejarum' in Malaysia, as well as the world. Our finding has broadened the distribution and host range of N. cubana , indicating that it poses potential damage to the medicinal plant Bunga Jejarum 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 Pečenka 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.

Mei (Prunus mume Sieb. et Zucc.), widely distributed in East Asian countries for both fruiting- and flowering-purposes, is susceptible to viral infections (Marais et al. 2018). Infection by plum bark necrosis stem pitting-associated virus (PBNSPaV) or little cherry virus 2 (LChV-2) possibly caused overall yield loss in mei in Japan due to incomplete flower development, low fruit bearing rate, and interveinal chlorosis (Numaguchi et al. 2019). Virus-like disease showing mosaic, interveinal chlorosis, vein clearing, or necrotic spot on leaf was observed in mei trees in Beijing, Wuhan, Wuxi, and Nanjing in spring and early summer from 2017 to 2018. Symptomatic leaves collected from the four regions were pooled as two samples for RNA-sequencing (RNA-seq) analysis. After ribosomal RNA (rRNA)-depletion, total RNA extracted by TRNzol reagent (TIANGEN, China) was subjected to library construction using NEBNext Ultra RNA Library Prep Kit (NEB, MA, USA) and sequenced on an Illumina Hiseq 4000 (Novogene, China). Sequencing data was filtered, screened, and assembled as described previously (Zhou et al. 2020) to generate contigs, following by BLAST-x/n search in viral genomes in GenBank. We identified >300 contigs (208-10756 nt) homologous to Asian prunus virus 1 and Asian prunus virus 2 (APV1 and 2), mume virus A (MuVA), PBNSPaV, and peach leaf pitting-associated virus (PLPaV), with 71-100% of nucleotide sequence identity values. APV1 and 2 have been reported in mei in China (Wang et al. 2018), here, we focused on the other three viruses. Contigs homologous to these three viruses were further assembled into three scaffolds of 14,224 nt, 1107 nt, and 753 nt for PBNSPaV, MuVA, and PLPaV, respectively. The scaffold of PBNSPaV (MW217574) nearly covered the whole genome of the isolate VIC3 from Australia (LC523039.1) (Kinoti et al. 2020) with 92.30% of sequence identity; the scalffold of MuVA (MW217572) covered 14.50% of the genome of the isolate pm14 from Japan (NC 040568.1) (Marais et al.2018) with 98.47% sequence identity; the scaffold of PLPaV (MW217573) covered 15.26% of the genome of the isolate XJ-6 from peach (KY867750.1) (He et al. 2017) with 85.23% sequence identity. Presence of the three viruses were verified by RT-PCR detection using designed specific primers for PBNSPaV (Forward: 5'-CAACAAAACTCCCACAGCGG-3 [positions 4014-4033, NC_009992.1] / Reverse: 5'-GCCAAAAGAAGTGCTGGTGG-3' [positions 4659-4640, NC_009992.1]), MuVA (Forward: 5'-AAGAGAATTACTTCAATGCCCTC-3' [positions 171-194, NC_040568.1] / Reverse: 5'-GATATCCAAGATACGATTAGCCAG-3' [positions 533-510, NC_040568.1]), and PLPaV (Forward: 5'-GCTATATCTCAACAACTGCAAGAA-3 [positions 5798-5821, KY867750.1] / Reverse: 5'- GAGTGATACATAGTCCACAGAGAT-3'[ positions 6045-6022, KY867750.1]). The amplified 626, 350 and 251 bp fragments of PBNSPaV, MuVA and PLPaV had 91.47%, 98.07% and 81.89% sequence identity to their respective reference sequences. This is the first report of PBNSPaV and MuVA infecting mei in China, and more importantly, the first report of a new host for PLPaV. In addition, 30 collected leaf samples from Nanjing and Wuhan were analyzed by RT-PCR and 15, 6, and 5 samples tested positive to PLPaV, PBNSPaV, and MuVA, respectively. Although it is difficult to link a particular virus with the observed symptoms due to mixed infections, the symptoms were significantly associated with viral infection because almost all symptomatic leaf samples were virus(es)-positive. Further studies would be required to determine the distribution and impact of these viruses on mei trees and other stone fruits species and to understand the possibility that mei trees may play a role in PLPaV epidemiology.