The oil palm industry, especially in Indonesia and Malaysia is being threatened by Basal Stem Rot (BSR) disease caused by Ganoderma boninense. There is no conclusive remedy in handling this disease effectively. In this study, metagenomics analysis of soil were analyzed for a better understanding of the microbial diversity in relation to BSR disease. Study was conducted in three plantation sites of Sabah, Malaysia which incorporated different disease management and agronomic practices. The estates are located at Sandakan (Kam Cheong Plantation), Lahad Datu (FGV Ladang Sahabat) and Tawau (Warisan Gagah). Soil samples were collected from disease free, high and low BSR incidence plots. Illumina MiSeq metagenomic analysis using V3-V4 region of 16S rRNA gene was employed to study the microbial diversity. Bacteria (97.4%) and Archaea (0.2%) were found majority in kingdom taxonomy level. The most abundant phyla were Proteobacteria, Acidobacteria, Actinobacteria, and Verrucomicrobia. Higher alpha diversity of all species was observed among all tested soil from each estates. Beta analysis was analyzed using non phylogenetic UnifRac matrix and visualized using Principal Coordinates Analysis (PCoA). The tested soil samples in Kam Cheong Plantation were found to have similar bacterial communities. The data provided is useful as an indicator in developing biology controls against Ganoderma boninense.
Ganoderma boninense (G. boninense) is a soil-borne fungus threatening oil palm at the present. It causes basal stem rot disease on oil palm. Within six months, this fungus can cause an oil palm plantation to suffer a significant 43% economic loss. The high persistence and nature of spread of G. boninense in soil make control of the disease challenging. Therefore, controlling the pathogen requires a thorough understanding of the mechanisms that underlie pathogenicity as well as its interactions with host plants. In this paper, we present the general characteristics, the pathogenic mechanisms, and the host's defensive system of G. boninense. We also review upcoming and most promising techniques for disease management that will have the least negative effects on the environment and natural resources.
Some species of Ganoderma, such as G. lucidum, are well-known as traditional Chinese medicine (TCM), and their pharmacological value was scientifically proven in modern days. However, G. boninense is recognized as an oil palm pathogen, and its biological activity is scarcely reported. Hence, this study aimed to investigate the antibacterial properties of G. boninense fruiting bodies, which formed by condensed mycelial, produced numerous and complex profiles of natural compounds. Extract was cleaned up with normal-phase SPE and its metabolites were analyzed using liquid chromatography-mass spectrometry (LCMS). From the disc diffusion and broth microdilution assays, strong susceptibility was observed in methicillin-resistant Staphylococcus aureus (MRSA) in elute fraction with zone inhibition of 41.08 ± 0.04 mm and MIC value of 0.078 mg mL-1. A total of 23 peaks were detected using MS, which were putatively identified based on their mass-to-charge ratio (m/z), and eight compounds, which include aristolochic acid, aminoimidazole ribotide, lysine sulfonamide 11v, carbocyclic puromycin, fenbendazole, acetylcaranine, tigecycline, and tamoxifen, were reported in earlier literature for their antimicrobial activity. Morphological observation via scanning electron microscope (SEM), cell membrane permeability, and integrity assessment suggest G. boninense extract induces irreversible damage to the cell membrane of MRSA, thus causing cellular lysis and death.
The microbial diseases cause significant damage in agriculture, resulting in major yield and quality losses. To control microbiological damage and promote plant growth, a number of chemical control agents such as pesticides, herbicides, and insecticides are available. However, the rising prevalence of chemical control agents has led to unintended consequences for agricultural quality, environmental devastation, and human health. Chemical agents are not naturally broken down by microbes and can be found in the soil and environment long after natural decomposition has occurred. As an alternative to chemical agents, biocontrol agents are employed to manage phytopathogens. Interest in lactic acid bacteria (LAB) research as another class of potentially useful bacteria against phytopathogens has increased in recent years. Due to the high level of biosafety, they possess and the processes they employ to stimulate plant growth, LAB is increasingly being recognized as a viable option. This paper will review the available information on the antagonistic and plant-promoting capabilities of LAB and its mechanisms of action as well as its limitation as BCA. This review aimed at underlining the benefits and inputs from LAB as potential alternatives to chemical usage in sustaining crop productivity.
Basal stem rot (BSR) disease on Elaeis guineens is known to be caused by members of the pathogenic fungal genus Ganoderma, especially the species Ganoderma boninense This species affects oil palm plantation in Sabah, Malaysia. The genome sequence (52.28 Mbp) will add to the representation of this genus, especially in regard to BSR disease.
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.
In the 1920s, Lewis Stadler initiated the introduction of permanent improvements to the genetic makeup of irradiated plants. Since then, studies related to breeding mutations have grown, as efforts have been made to expand and improve crop productivity and quality. Stadler's discovery began with x-rays on corn and barley and later extended to the use of gamma-rays, thermal, and fast neutrons in crops. Radiation has since been shown to be an effective and unique method for increasing the genetic variability of species, including rice. Numerous systematic reviews have been conducted on the impact of physical mutagens on the production and grain quality of rice in Southeast Asia. However, the existing literature still lacks information on the type of radiation used, the rice planting materials used, the dosage of physical mutagens, and the differences in mutated characteristics. Therefore, this article aims to review existing literature on the use of physical mutagens in rice crops in Southeast Asian countries. Guided by the PRISMA Statement review method, 28 primary studies were identified through a systematic review of the Scopus, Science Direct, Emerald Insight, Multidisciplinary Digital Publishing, and MDPI journal databases published between 2016 and 2020. The results show that 96% of the articles used seeds as planting materials, and 80% of the articles focused on gamma-rays as a source of physical mutagens. The optimal dosage of gamma-rays applied was around 100 to 250 Gy to improve plant development, abiotic stress, biochemical properties, and nutritional and industrial quality of rice.
Open pancreaticoduodenectomy, also known as Whipple surgery, is a complex and painful procedure that requires a multi-modal analgesic approach for successful post-operative rehabilitation and recovery. While thoracic epidural analgesia (TEA) remains the gold standard for pain relief after open upper abdominal surgery, it carries many risks that may outweigh the potential benefits of the technique. Furthermore, in laparoscopic converted to open pancreaticoduodenectomy cases, post-operative placement of a thoracic epidural catheter is inconvenient to the patient due to pain and positioning. The external oblique intercostal (EOI) block is a novel method that provides somatic analgesia to the upper abdomen. We present a case of bilateral EOI block with catheter insertion for post-operative analgesia in a patient who underwent laparoscopic converted to open Whipple surgery.
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.
'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.
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.
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.
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.
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.
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 β-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.
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 β-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.
'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 β-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.