First Report of Athelia rolfsii Causing Damping-Off and Leaf Blight on Basella alba in Malaysia.

Basella alba is an evergreen perennial vine that grows as an edible leafy vegetable in Malaysia (Nordin et al. 2007). During January 2021, a cottony white hypha associated with aggregates of white to brown sclerotia and symptoms of damping-off were visualized on the stem base of B. alba at the soil surface in an isolated field (~0.03 ha) located in the district of Penampang, Sabah province, Malaysia (5°56'51.0"N 116°04'31.8"E). Yellowing and wilting of leaves, and defoliation were observed after four days of the development of damping-off. Survey was conducted on 100 plants which 30 were found infected. The disease severity (90%) on stems was estimated using IMAGEJ. Symptomatic stem tissues were surface sterilized with 75% of ethanol for 1 min, washed with 2% of sodium hypochlorite solution for 1 min, rinsed thrice with sterile distilled water, air dried and plated on potato dextrose agar (PDA). Plates were incubated for 7 days at 25°C in the dark. After 7 days, fungi were isolated; colony color was white and had a cottony appearance. On day 14, white to brown sclerotia 1.0 to 2.2 mm in diameter were produced. Hyaline septate hyphae with clamp connections and multiple nuclei were seen. Conidia and conidiophores were absent from the colony on PDA. Genomic DNA of fungi was extracted based on Khoo et al. (2022a and 2022b). PCR amplification (Khoo et al. 2022b) was performed using primer set ITS1/ITS4, EF983/EF2218 and LR0R/LR05 to amplify the internal transcribed spacer (ITS) region of rDNA, partial translation elongation factor 1 alpha (TEF-1α) gene and partial large subunit ribosomal RNA (LSU rRNA) gene, respectively (Vilgalys and Hester 1990; White et al. 1990; Carbone and Kohn, 1999; Rehner 2001). Phylogenetic analysis indicated that the isolates formed a supported clade to the related Athelia rolfsii sequences. The sequencing result (GenBank Accession Nos. OK465460, OP809607, OP857217) had a 99% identity over 625 bp, 941 bp, and 1,101 bp with the corresponding gene sequence of A. rolfsii (GenBank Accession Nos. MN622806, AY635773, MW322687) after analysis in BLASTn program. Pathogenicity test was performed based on Le (2011). Three 8-week-old B. alba plants cultivated on sterilized soil were inoculated with 5-mm mycelia plugs from 7-day-old culture. A plug was put on the upper soil surface layer 2 cm away from the base of the stem of B. alba plant before fully covered with a layer of sterilized soil. Plants that were inoculated with sterile PDA plugs served as the control treatment. Plastic bags were used to cover the plants after inoculation for 24 h before keeping them in a glasshouse under ambient conditions. Water-soaked and brown lesions were visualized on the stem base of all inoculated plants after four days of inoculation. Symptom of damping-off and leaf blight was observed after another 3 days. No symptoms developed on the mock controls. The pathogenicity test was repeated twice. Re-isolation was performed from the symptomatic tissues of inoculated plants and mock controls. The isolates reisolated from the symptomatic tissues were verified as A. rolfsii based on morphology and molecular characterization, thus fulfilling Koch's postulates. No pathogens were isolated from the mock controls. To our knowledge, this is the first report of A. rolfsii causing damping-off and leaf blight on B. alba in Malaysia, as well as worldwide. Our findings documented the wider geographical and host range of A. rolfsii and indicate its potential threat to B. alba production in Malaysia.

First report of Colletotrichum siamense Causing anthracnose on Cinnamomum camphora in Malaysia.

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 (106 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.

First report of Colletotrichum siamense Causing Anthracnose on Crinum asiaticum 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 (106 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.

First Report of Neopestalotiopsis cubana Causing leaf blight on Ixora chinensis 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. cubanabased 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 × 106 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.

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.