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.

First Report of Curvularia lunata Causing Leaf Spot on Oryza sativa in Sabah, Malaysian Borneo.

Rice (Oryza sativa L.) has been farmed in Malaysia since ancient times and is one of the most important commercial crops (Ma'arup et al. 2020). Throughout January to August 2022, chlorotic spots with brown halos ranging 2 to 10 mm wide were found on upper leaves of rice variety Mahsuri in the vegetative stage with a severity and incidence of approximately 60% and 100%, respectively in Kampung Tagas, Sabah, Malaysian Borneo (06°09'41.8"N, 116°13'45.1"E). As the disease developed, the spots coalesced into larger chlorotic spots. Three leaf pieces (5 x 5 mm) were excised from lesion margins, surface sterilized based on Khoo et al. (2022a), before plating on water agar (WA) at 25°C. Purification of fungi was conducted on WA using hyphal tip isolation. When three pure cultures were obtained, the fungi were cultured on potato dextrose agar (PDA) and WA for 7 days in 12 h light and 12 h dark at 25°C for the macro- and micro-morphological characterization, respectively. The colonies of the three isolates on PDA were initially gray, later becoming dark. Conidia (n=30) were fusiform, smooth-walled, dark-brown, and melanized with three transverse septa, measuring 7.3 to 11.4 × 16.2 to 27.2 µm. The isolates were named Tagas01, Tagas02, Tagas03. Genomic DNA was extracted from fresh mycelia of the pathogens based on the extraction method described by Khoo et al. (2022b). The primers ITS1/ITS4 (White et al. 1990), GPD1/GPD2 (Berbee et al. 1991), and EF1-983F/EF1-2218R (Schochet al. 2009) were used to amplify the internal transcribed spacer (ITS) region of rDNA, partial fragments of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor (EF-1α) region, respectively based on PCR conditions as described previously (Khoo et al. 2022a). The sequences were deposited in GenBank under accession numbers OP268402, OP271304, OP271305 (677/677 bp) (ITS), OP270699, OP270703, OP270704 (609/613 bp) (GAPDH), OP270700-OP270702 (928/930 bp) (EF-1α). They were 99.35-100% similar to the Curvularia lunata ITS (HF934911), GAPDH (LT715821), and Curvularia dactyloctenicola EF-1α (MF490858) type sequences. Although C. dactyloctenicolais related to C. lunata, the conidia of the former are much wider making them easier to differentiate (Marin-Felix et al. 2017). Phylogenetic analysis using maximum likelihood based on the combined ITS, GAPDH and EF-1α sequences indicated that the isolate formed a supported clade to C. lunata. The pathogens were identified as C. lunata based on morphological and molecular characterization. Koch's postulates were performed. Three replicate healthy rice at the vegetative stage were sprayed with a spore suspension of 1 × 106 spore/ml in distilled sterilized water, prepared from 1-week-old fungal culture, grown in the dark on WA. Three replicate rice plants were sprayed with distilled sterilized water as control. Plants were covered with transparent polyethylene bags to keep moisture, and kept in a greenhouse at ~27°C. Bags were removed after 4 days of incubation. Monitoring and incubation were performed in greenhouse based on Iftikhar et al. (2022). The pathogenicity test was also performed using isolate Tagas02 and Tagas03. All inoculated leaves developed symptoms as described after 6 days post-inoculation, whereas no symptoms occurred on controls. The experiments were repeated twice. The reisolated fungi were identical to the pathogen morphologically and molecularly, thus fulfilling Koch's postulates. C. lunata has been reported in Peninsular Malaysia (Lee et al. 2012). This is the first report of C. lunata causing leaf spot on Oryza sativa in Sabah, Malaysian Borneo. This illness not only reduces yields and lowers milling quality, but it may also be mistaken for rice blast, necessitating needless fungicide spraying.

Fucoxanthin Production of Microalgae under Different Culture Factors: A Systematic Review.

Fucoxanthin is one of the light-harvesting pigments in brown microalgae, which is increasingly gaining attention due to its numerous health-promoting properties. Currently, the production of microalgal fucoxanthin is not yet feasible from an economic perspective. However, the cultivation of microalgae at favourable conditions holds great potential to increase the viability of this fucoxanthin source. Hence, this study aimed to review the fucoxanthin production of microalgae under different conditions systematically. A literature search was performed using the Web of Science, Scopus and PubMed databases. A total of 188 articles were downloaded and 28 articles were selected for the current review by two independent authors. Microalgae appeared to be a more reliable fucoxanthin source compared to macroalgae. Overall, a consensus fucoxanthin production condition was obtained and proposed: light intensity ranging from 10 to 100 µmol/m2/s could achieve a higher fucoxanthin content. However, the optimal light condition in producing fucoxanthin is species-specific. The current review serves as an antecedent by offering insights into the fucoxanthin-producing microalgae response to different culture factors via a systematic analysis. With the current findings and recommendations, the feasibility of producing fucoxanthin commercially could be enhanced and possibly achieve practical and sustainable fucoxanthin production.

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 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 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.

A Review on a Hidden Gem: Phycoerythrin from Blue-Green Algae.

Phycoerythrin (PE) is a pink/red-colored pigment found in rhodophytes, cryptophytes, and blue-green algae (cyanobacteria). The interest in PE is emerging from its role in delivering health benefits. Unfortunately, the current cyanobacterial-PE (C-PE) knowledge is still in the infant stage. It is essential to acquire a more comprehensive understanding of C-PE. This study aimed to review the C-PE structure, up and downstream processes of C-PE, application of C-PE, and strategies to enhance its stability and market value. In addition, this study also presented a strengths, weaknesses, opportunities, and threats (SWOT) analysis on C-PE. Cyanobacteria appeared to be the more promising PE producers compared to rhodophytes, cryptophytes, and macroalgae. Green/blue light is preferred to accumulate higher PE content in cyanobacteria. Currently, the prominent C-PE extraction method is repeated freezing-thawing. A combination of precipitation and chromatography approaches is proposed to obtain greater purity of C-PE. C-PE has been widely exploited in various fields, such as nutraceuticals, pharmaceuticals, therapeutics, cosmetics, biotechnology, food, and feed, owing to its bioactivities and fluorescent properties. This review provides insight into the state-of-art nature of C-PE and advances a step further in commercializing this prospective pigment.

Uncovering Research Trends of Phycobiliproteins Using Bibliometric Approach.

Phycobiliproteins are gaining popularity as long-term, high-value natural products which can be alternatives to synthetic products. This study analyzed research trends of phycobiliproteins from 1909 to 2020 using a bibliometric approach based on the Scopus database. The current findings showed that phycobiliprotein is a burgeoning field in terms of publications outputs with "biochemistry, genetics, and molecular biology" as the most related and focused subject. The Journal of Applied Phycology was the most productive journal in publishing articles on phycobiliproteins. Although the United States of America (U.S.A.) contributed the most publications on phycobiliproteins, the Chinese Academy of Sciences (China) is the institution with the largest number of publications. The most productive author on phycobiliproteins was Glazer, Alexander N. (U.S.A.). The U.S.A. and Germany were at the forefront of international collaboration in this field. According to the keyword analysis, the most explored theme was the optimization of microalgae culture parameters and phycobiliproteins extraction methods. The bioactivity properties and extraction of phycobiliproteins were identified as future research priorities. Synechococcus and Arthrospira were the most cited genera. This study serves as an initial step in fortifying the phycobiliproteins market, which is expected to exponentially expand in the future. Moreover, further research and global collaboration are necessary to commercialize phycobiliproteins and increase the consumer acceptability of the pigments and their products.

The Critical Studies of Fucoxanthin Research Trends from 1928 to June 2021: A Bibliometric Review.

Fucoxanthin is a major carotenoid in brown macroalgae and diatoms that possesses a broad spectrum of health benefits. This review evaluated the research trends of the fucoxanthin field from 1928 to June 2021 using the bibliometric method. The present findings unraveled that the fucoxanthin field has grown quickly in recent years with a total of 2080 publications. Japan was the most active country in producing fucoxanthin publications. Three Japan institutes were listed in the top ten productive institutions, with Hokkaido University being the most prominent institutional contributor in publishing fucoxanthin articles. The most relevant subject area on fucoxanthin was the agricultural and biological sciences category, while most fucoxanthin articles were published in Marine Drugs. A total of four research concepts emerged based on the bibliometric keywords analysis: "bioactivities", "photosynthesis", "optimization of process'', and "environment". The "bioactivities" of fucoxanthin was identified as the priority in future research. The current analysis highlighted the importance of collaboration and suggested that global collaboration could be the key to valorizing and efficiently boosting the consumer acceptability of fucoxanthin. The present bibliometric analysis offers valuable insights into the research trends of fucoxanthin to construct a better future development of this treasurable carotenoid.

A Recommendation for a Pre-Standardized Marine Microalgal Dry Weight Determination Protocol for Laboratory Scale Culture Using Ammonium Formate as a Washing Agent.

Microalgal biomass is one of the crucial criteria in microalgal studies. Many reported methods, even the well-established protocol on microalgal dry weight (DW) determination, vary greatly, and reliable comparative assessment amongst published results could be problematic. This study aimed to determine the best condition of critical parameters in marine microalgal DW determination for laboratory-scale culture using four different marine microalgal species. These parameters included the washing process, grades of glass microfiber filter (GMF), GMF pretreatment conditions, washing agent (ammonium formate) concentrations, culture: washing agent ratios (v:v) and washing cycles. GMF grade GF/A with precombustion at 450 °C provided the most satisfactory DW and the highest ash-free dry weight (AFDW)/DW ratio. Furthermore, 0.05 M ammonium formate with 1:2 culture: washing agent ratio and a minimum of two washing cycles appeared to be the best settings of microalgal DW determination. The present treatment increased the AFDW/DW ratio of the four respective microalgae by a minimum of 19%. The findings of this study could serve as a pivotal reference in developing a standardized protocol of marine microalgal DW determination to obtain veracious and reliable marine microalgal DW.

Tuning the Electronic Structures of Multimetal Oxide Nanoplates to Realize Favorable Adsorption Energies of Oxygenated Intermediates.

Highly active oxygen evolution reaction (OER) electrocatalysts are important to effectively transform renewable electricity to fuel and chemicals. In this work, we construct a series of multimetal oxide nanoplate OER electrocatalysts through successive cation exchange followed by electrochemical oxidation, whose electronic structure and diversified metal active sites can be engineered via the mutual synergy among multiple metal species. Among the examined multimetal oxide nanoplates, CoCeNiFeZnCuOx nanoplates exhibit the optimal adsorption energy of OER intermediates. Together with the high electrochemical active surface area, the CoCeNiFeZnCuOx nanoplates manage to deliver a small overpotential of 211 mV at an OER current density of 10 mA cm-2 (η10) with a Tafel slope as low as 21 mV dec-1 in 1 M KOH solution, superior to commercial IrO2 (339 mV at η10, Tafel slope of 55 mV dec-1), which can be stably operated at 10 mA cm-2 (at an overpotential of 211 mV) and 100 mA cm-2 (at an overpotential of 307 mV) for 100 h.

Optimization of the Freezing-Thawing Method for Extracting Phycobiliproteins from Arthrospira sp.

The freezing-thawing method had been reported to be the best phycobiliprotein extraction technique. However, optimum parameters of this extraction method for Arthrospira sp. (one of the major phycobiliprotein sources) still remained unclear. Hence, this study aimed to optimize the freezing-thawing parameters of phycobiliprotein extraction in Arthrospira sp. (UPMC-A0087). The optimization of the freezing-thawing method was conducted using different solvents, biomass/solvent ratios, temperatures, time intervals and freezing-thawing cycles. The extracted phycobiliproteins were quantified using a spectrophotometric assay. Double distilled water (pH 7) with a 0.50% w/v biomass/solvent ratio was the most efficient solvent in extracting high concentrations and purity of phycobiliproteins from Arthrospira sp. In addition, the combination of freezing at -80 °C (2 h) and thawing at 25 °C (24 h) appeared to be the optimum temperature and extraction time to obtain the highest amount of phycobiliproteins. A minimum of one cycle of freezing and thawing was sufficient for extracting high concentrations of phycobiliproteins. The findings from this study could reduce the cost and labor needed for extracting high quality phycobiliproteins. It also allowed the harvesting of large amounts of valuable phycobiliproteins.

Hydrophilic Nitrogen and Sulfur Co-doped Molybdenum Carbide Nanosheets for Electrochemical Hydrogen Evolution.

Nitrogen and sulfur dual-doped Mo2 C nanosheets provide low operating potential (-86 mV for driving 10 mA cm(-2) of current density). Co-doping of N and S heteroatoms can improve the wetting property of the Mo2C electrocatalyst in aqueous solution and induce synergistic effects via σ-donation and π-back donation with hydronium cation.