Molecular and aflatoxigenicity analyses of Aspergillus flavus isolates indigenous to grain corn in Malaysia; potentials for biological control.

AIMS: The present work aimed to distinguish the indigenous Aspergillus flavus isolates obtained from the first (pioneer) grain corn farms in Terengganu, Malaysia, into aflatoxigenic and non-aflatoxigenic by molecular and aflatoxigenicity analyses, and determine the antagonistic capability of the non-aflatoxigenic isolates against aflatoxigenic counterparts and their aflatoxin production in vitro. METHODS AND RESULTS: Seven A. flavus isolates previously obtained from the farms were characterized molecularly and chemically. All isolates were examined for the presence of seven aflatoxin biosynthesis genes, and their aflatoxigenicity was confirmed using high performance liquid chromatography with fluorescence detector. Phylogenetic relationships of all isolates were tested using ITS and ß-tubulin genes. Of the seven isolates, two were non-aflatoxigenic, while the remaining were aflatoxigenic based on the presence of all aflatoxin biosynthesis genes tested and the productions of aflatoxins B1 and B2. All isolates were also confirmed as A. flavus following phylogenetic analysis. The indigenous non-aflatoxigenic isolates were further examined for their antagonistic potential against aflatoxigenic isolates on 3% grain corn agar. Both non-aflatoxigenic isolates significantly reduced AFB1 production of the aflatoxigenic isolates. CONCLUSION: The indigenous non-aflatoxigenic A. flavus strains identified in the present work were effective in controlling the aflatoxin production by the aflatoxigenic A. flavus isolates in vitro and can be utilized for in situ testing.

Piper sarmentosum Roxb. Inhibits Angiotensin-Converting Enzyme Activity in Phorbol 12-Myristate-13-Acetate-Induced Endothelial Cells.

Angiotensin-converting enzyme (ACE) plays a crucial role in the pathogenesis of hypertension. Piper sarmentosum Roxb., an herb known for its antihypertensive effect, lacks a comprehensive understanding of the mechanism underlying its antihypertensive action. This study aimed to elucidate the antihypertensive mechanism of aqueous extract of P. sarmentosum leaves (AEPS) via its modulation of the ACE pathway in phorbol 12-myristate-13-acetate (PMA)-induced human umbilical vein endothelial cells (HUVECs). HUVECs were divided into five groups: control, treatment with 200 µg/mL AEPS, induction 200 nM PMA, concomitant treatment with 200 nM PMA and 200 µg/mL AEPS, and treatment with 200 nM PMA and 0.06 µM captopril. Subsequently, ACE mRNA expression, protein level and activity, angiotensin II (Ang II) levels, and angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) mRNA expression in HUVECs were determined. AEPS successfully inhibited ACE mRNA expression, protein and activity, and angiotensin II levels in PMA-induced HUVECs. Additionally, AT1R expression was downregulated, whereas AT2R expression was upregulated. In conclusion, AEPS reduces the levels of ACE mRNA, protein and activity, Ang II, and AT1R expression in PMA-induced HUVECs. Thus, AEPS has the potential to be developed as an ACE inhibitor in the future.

Modified-Malay Eating Behavior and Pattern Questionnaire (Malay-EBPQ): translation and validation among Malaysian women.

BACKGROUND: Eating behavior primarily depends on eating patterns which are largely influenced by interactions between physiology, environment, psychology, culture and socio-economic status. This study was designed to translate and validate the Eating Behavior Pattern Questionnaire (EBPQ) among Malaysian women. METHODS: A cross-sectional study involving translation and validation of the English version of EBPQ. The original questionnaire, contained 51 items extracted into six domains was translated in Malay using forward and backward translation, pre-tested and validated among conveniently sampled female healthcare personnel. Vegetarians, pregnant ladies and women in confinement were excluded due to special daily dietary plans. Construct validity, reliability and feasibility were analyzed using Exploratory Factor Analysis (EFA) and Confirmatory Factor Analysis (CFA). RESULTS: During translation, item modifications were made and subjected to field testing among 394 women. The original questionnaire was used as a reference to identify the positioning of items in constructs. Fifteen items were removed due to poor correlation with items within constructs. Seven factors were extracted using Varimax rotation with Kaiser-Meyer-Olkin (KMO) value range from 0.725-0.872 and significant Bartlett's test of Sphericity (p < 0.001). The item-loading of the items within the constructs ranged between 0.415-0.812 (explained variation = 62.7%). Cultural and lifestyle behavior was relabeled to lifestyle and behavioral eating, and snacking on sweets was relabeled as snacking pattern. Emotional eating was divided into two sub-factors as snacking behavior and emotional influence. CFA resulted with an acceptable fit with no presence of floor and ceiling effects. Intra-class correlation coefficient (ICC) for all the constructs were reported good and excellent. The overall internal consistency was reported as good. CONCLUSION: The modified 36-item Malay-EBPQ had moderate internal consistency, reliable and fit with multi-dimensional measures of eating behaviors and dietary patterns among women in the multi-racial population with cultural diversity.

High referral accuracy for oral cancers and oral potentially malignant disorders using telemedicine.

OBJECTIVE: To evaluate the accuracy of MeMoSA®, a mobile phone application to review images of oral lesions in identifying oral cancers and oral potentially malignant disorders requiring referral. SUBJECTS AND METHODS: A prospective study of 355 participants, including 280 with oral lesions/variants was conducted. Adults aged ≥18 treated at tertiary referral centres were included. Images of the oral cavity were taken using MeMoSA®. The identification of the presence of lesion/variant and referral decision made using MeMoSA® were compared to clinical oral examination, using kappa statistics for intra-rater agreement. Sensitivity, specificity, concordance and F1 score were computed. Images were reviewed by an off-site specialist and inter-rater agreement was evaluated. Images from sequential clinical visits were compared to evaluate observable changes in the lesions. RESULTS: Kappa values comparing MeMoSA® with clinical oral examination in detecting a lesion and referral decision was 0.604 and 0.892, respectively. Sensitivity and specificity for referral decision were 94.0% and 95.5%. Concordance and F1 score were 94.9% and 93.3%, respectively. Inter-rater agreement for a referral decision was 0.825. Progression or regression of lesions were systematically documented using MeMoSA®. CONCLUSION: Referral decisions made through MeMoSA® is highly comparable to clinical examination demonstrating it is a reliable telemedicine tool to facilitate the identification of high-risk lesions for early management.

First report of Pantoea anthophila causing shoot dieback disease of Ceylon ironwood (Mesua ferrea Linn.) in Malaysia.

Ceylon ironwood (Mesua ferrea Linn.) or Penaga lilin is one of Asia's most popular tropical herbal plants, including Malaysia (Sharma et al., 2017). The trees are cultivated for their aesthetic value and pharmacological properties, especially as traditional remedies for asthma, dermatopathy, inflammation, and rheumatic conditions (Adib et al., 2019). In August 2022, a disease survey was conducted on Ceylon ironwood trees ranging from 5 to 12 years old in Botanical Park, Putrajaya, Malaysia, with 80% exhibiting shoot dieback disease of the 15 trees exhibiting shoot dieback disease. Symptoms include irregular, water-soaked with brown lesions on young leaves and shoots, where the small lesion coalesced and formed broad necrotic regions, subsequently causing dieback and gradual defoliation. Three infected shoots were collected from each tree, excised into small pieces (10 to 20 mm), immersed with 75% ethanol for 3 min, washed with 2% NaOCl solution for 1 min, and rinsed twice for 1 min in sterilized distilled water. A 10 µl aliquot of the sample suspension was streaked onto nutrient agar (NA) and incubated for 24 h to 48 h at 35 °C. A total of 15 isolates with similar morphology were obtained, and each isolate was re-streaked three times to obtain pure colonies that were round, smooth, with irregular edges, and produced yellow pigment in culture. All isolates were Gram-negative, negative for indole production, and utilized glucose, maltose, trehalose, sucrose, D-lactose, and pectin. Three representative isolates (C001, C002, and C003) with similar morphology were selected for further characterization. The total genomic DNA of all isolates was extracted from overnight cultures using Geneaid™ DNA Isolation Kit (Geneaid Biotech Ltd., Taiwan). PCR amplification of 16S rDNA (Zhou et al., 2015) and species-specific infB (Brady et al., 2008) genes was performed, and each of the ~1500 bp and ~900 bp amplicons were sequenced. BLASTn and phylogenetic analyses revealed all isolates were 100% identical to Pantoea anthophila (P. anthophila) LGM 2558 strains (Accession Nos. NR_116749 and NR_116113) for the 16S rDNA gene. They were 99% identical to P. anthophila CL1 strain (Accession Number CP110473) for infB gene. These sequences were later deposited in the GenBank (Accession Nos. OQ772233, OQ772234, and OQ772235 for 16S rDNA gene, and OQ803527, OQ803528, and OQ803529 for infB gene). For the pathogenicity test, healthy Ceylon ironwood seedlings' shoots were inoculated with 10 mL of each isolate suspension (1 x 108 CFU/ml) by spraying the inoculum on the young shoots using a sterilized spray bottle. Control seedlings were inoculated with sterile water. The inoculated shoots were covered with a sealed plastic bag to maintain the moisture and were kept in the greenhouse with temperatures ranging from 26 to 35 °C. The experiments were repeated twice, with three replicates for each treatment. Inoculated shoots showed dieback symptoms like natural infection, including irregular, water-soaked, and brown lesions on leaves and young shoots at 10 days post-inoculation. Control seedlings remained asymptomatic. The pathogen was re-isolated and identified via sequencing of the 16S rDNA and infB genes, thus fulfilling Koch's postulates. Previously, P. anthophila has been reported to cause soft rot in wampee plants in China (Zhou et al., 2015) and leaf blight of cotton in Pakistan (Tufail et al., 2020). To our knowledge, this is the first report of P. anthophila causing shoot dieback disease of Ceylon ironwood trees in Malaysia. Plant disease management strategies need to be established to reduce losses due to P. anthophila infection since the pathogen could limit Ceylon ironwood tree production in Malaysia.

A laudable strategy to manage bacterial panicle blight disease of rice using biocontrol agents.

Bacterial panicle blight (BPB) disease is a dreadful disease in rice-producing countries. Burkholderia glumae, a Gram-negative, rod-shaped, and flagellated bacterium was identified as the primary culprit for BPB disease. In 2019, the disease was reported in 18 countries, and to date, it has been spotted in 26 countries. Rice yield has been reduced by up to 75% worldwide due to this disease. Interestingly, the biocontrol strategy offers a promising alternative to manage BPB disease. This review summarizes the management status of BPB disease using biological control agents (BCA). Bacteria from the genera Bacillus, Burkholderia, Enterobacter, Pantoea, Pseudomonas, and Streptomyces have been examined as BCA under in vitro, glasshouse, and field conditions. Besides bacteria, bacteriophages have also been reported to reduce BPB pathogens under in vitro and glasshouse conditions. Here, the overview of the mechanisms of bacteria and bacteriophages in controlling BPB pathogens is addressed. The applications of BCA using various delivery methods could effectively manage BPB disease to benefit the agroecosystems and food security.

First report of Burkholderia gladioli causing bacterial panicle blight of rice in Malaysia.

Rice (Oryza sativa) is a staple food for most of the world's populations, particularly in Asia (Gumma et al. 2011). The rice sector provides Malaysians with a food supply, food sufficiency, and income for growers (Man et al. 2009). From January to February 2022, panicle samples showing symptoms of bacterial panicle blight (BPB) disease, including reddish-brown, linear lesions with indistinct margins on flag-leaf sheaths and blighted, upright, grayish straw-colored florets with sterile and aborted grains on panicles were collected in granary areas in Sekinchan, Selangor, Malaysia with 90% disease incidence in fields. Surface-sterilization of infected leaf tissue was performed using 75% ethanol and 1% sodium hypochlorite, followed by rinsing three times in sterilized water. Leaf tissue was macerated in sterilized water and aliquots were spread on King's B agar medium, then cultured for 24 h to 48 h at 35 °C. All isolated bacteria were Gram-negative rods, positive for catalase and gelatinase but negative for indole, oxidase and hydrogen sulfide (H2S), and utilized sucrose, inositol, mannitol, glucose, and citrate. Colonies were circular and smooth-margined, producing a diffusible yellowish-green pigment on King's B agar medium, which are characteristics of Burkholderia species (Keith et al. 2005). Five representative isolates (UPMBG7, UPMBG8, UPMBG9, UPMBG15, UPMBG17) were selected for molecular and pathogenicity tests. PCR using specific primers targeting the gyrB gene for molecular characterization was performed, and the ∼470 bp amplicons were sequenced (Maeda et al. 2006) and deposited in GenBank (OM824438 to OM824442). A BLASTn analysis revealed that the five isolates were 99% identical to the B. gladioli reference strains MAFF 302533, GRBB15041, and LMG19584 in GenBank (AB190628, KX638432, and AB220898). A phylogenetic tree using Maximum-likelihood analysis of the gyrB gene sequences showed that the five isolates were 99% identical to B. gladioli reference strains (AB190628, KX638432, and AB220898). To verify the identification of these isolates, the 16S rDNA gene was amplified using 16SF/16SR primers (Ramachandran et al. 2021), producing ~1,400 bp amplicons. The resulting sequences of the five isolates (OM869953 to OM869957) were 98% identical to the reference strains of B. gladioli (NR113629 and NR117553). To confirm pathogenicity, 10 ml suspensions of the five isolates at of 108 CFU/ml were inoculated into the panicles and crowns of 75-day-old rice seedlings of local rice varieties MR269 and MR219 grown in a glasshouse with temperatures ranging from 37 °C to 41 °C (Nandakumar et al. 2009). Control rice seedlings were inoculated with sterilized water. All isolates produced BPB disease symptoms like those originally found in the rice fields at four weeks after inoculation. Control seedlings remained asymptomatic. To fulfill Koch's postulates, the bacteria were reisolated from symptomatic panicles and were confirmed as B. gladioli by sequence analysis of the gyrB and 16S rDNA genes. To our knowledge, this is the first report of B. gladioli causing BPB disease of rice in Malaysia. Since BPB disease causes a significant threat to the rice industry, it is crucial to investigate the diversity of this destructive pathogen for effective disease management strategies in Malaysia.

Recent Advances in Non-Invasive Blood Pressure Monitoring and Prediction Using a Machine Learning Approach.

Blood pressure (BP) monitoring can be performed either invasively via arterial catheterization or non-invasively through a cuff sphygmomanometer. However, for conscious individuals, traditional cuff-based BP monitoring devices are often uncomfortable, intermittent, and impractical for frequent measurements. Continuous and non-invasive BP (NIBP) monitoring is currently gaining attention in the human health monitoring area due to its promising potentials in assessing the health status of an individual, enabled by machine learning (ML), for various purposes such as early prediction of disease and intervention treatment. This review presents the development of a non-invasive BP measuring tool called sphygmomanometer in brief, summarizes state-of-the-art NIBP sensors, and identifies extended works on continuous NIBP monitoring using commercial devices. Moreover, the NIBP predictive techniques including pulse arrival time, pulse transit time, pulse wave velocity, and ML are elaborated on the basis of bio-signals acquisition from these sensors. Additionally, the different BP values (systolic BP, diastolic BP, mean arterial pressure) of the various ML models adopted in several reported studies are compared in terms of the international validation standards developed by the Advancement of Medical Instrumentation (AAMI) and the British Hypertension Society (BHS) for clinically-approved BP monitors. Finally, several challenges and possible solutions for the implementation and realization of continuous NIBP technology are addressed.

Prognostic Abilities of Pre- and Post-Treatment Inflammatory Markers in Oral Squamous Cell Carcinoma: Stepwise Modelling.

Background and Objectives: Studies examining the importance of inflammatory markers before treatment as prognosticators of OSCC are available, but information on post-therapy inflammatory markers and their prognostic significance is limited. This study aimed to evaluate the prognostic abilities of pre- and post-treatment inflammatory markers in patients with OSCC. Materials and Methods: In this retrospective analysis, information on 151 OSCC patients' socio-demographic, clinico-pathological, recurrence, metastasis, and survival data were gathered from clinical records. A multivariable Cox proportional hazards regression (stepwise model) was conducted to identify the prognostic predictors of OS and DFS. The multivariable models' performances were evaluated using Harrell's concordance statistics. Results: For OS, high pre-treatment LMR (HR 3.06, 95%CI 1.56, 5.99), and high post-treatment PLC (HR 3.35, 95%CI 1.71, 6.54) and PLR (HR 5.26, 95%CI 2.62, 10.58) were indicative of a poor prognosis. For DFS, high pre-treatment SII (HR 2.59, 95%CI 1.50, 4.48) and high post-treatment PLC (HR 1.92, 95%CI 1.11, 3.32) and PLR (HR 3.44, 95%CI 1.98, 5.07) were associated with increased mortality. The fitness of the OS and DFS stepwise Cox regression models were proven with a time-dependent AUC of 0.8787 and 0.8502, respectively. Conclusions: High pre-treatment levels of LMR and SII and high post-treatment levels of PLC and PLR are independent predictors of a poor prognosis for patients with OSCC.

Population data and genetic characteristics of 12 X-STR loci using the Investigator® Argus X-12 Quality Sensor kit for the Kedayan population of Borneo in Malaysia.

DNA profiling of X-chromosomal short tandem repeats (X-STR) has exceptional value in criminal investigations, especially for complex kinship and incest cases. In this study, Investigator® Argus X-12 Quality Sensor (QS) kits were successfully used to characterize 12 X-STR loci in 199 unrelated healthy Kedayan individuals living in Sabah and Sarawak, Malaysia. The LG1 haplogroup (DXS8378 - DXS10135 - DXS10148) has the largest HD (0.9799) as compared with all other closely linked haplotype groups examined (LG2; DXS7132-DXS10074-DXS10079, LG3; DXS10103-DXS10101-HPRTB and LG4; DXS10134-DXS7423-DXS10146). Data from statistical analysis showed that high combined of PDM, PDF, MEC_Krüger, MEC_Kishida, MEC_Desmarais, and MEC_Desmarais_duo values (0.999999994405922, 0.99999999999999, 0.999990463834938, 0.999999975914808, 0.999999975985006, and 0.999996491927194, respectively) in the Kedayan. In a two-dimensional scaling (MDS) plot and dendrogram constructed using allele frequencies at the 12 X-STR loci, Kedayan appear to be most closely related to their other Austronesian populations including the Malays and Filipinos as compared with other reference population groups. Findings from the present study thus demonstrate high genetic variability across the 12 tested X-STR loci and can be used for population studies and forensic applications.

First Report of Lasiodiplodia theobromae Causing Postharvest Fruit Rot on Guava (Psidium guajava) in Malaysia.

Guava (Psidium guajava L.) is an economically important tropical fruit crop and is cultivated extensively in Malaysia. In September and October 2019, postharvest fruit rot symptoms were observed on 30% to 40% of guava fruit cv. Kampuchea in fruit markets of Puchong and Ipoh cities in the states of Selangor and Perak, Malaysia. Initial symptoms appeared as brown, irregular, water-soaked lesions on the upper portion of the fruit where it was attached to the peduncle. Subsequently, lesions then progressed to cover the whole fruit (Fig.1A). Lesions were covered with an abundance of black pycnidia and grayish mycelium. Ten symptomatic guava fruit were randomly collected from two local markets for our investigation. For fungal isolation, small fragments (5×5 mm) were excised from the lesion margin, surface sterilized with 0.5% NaOCl for 2 min, rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA) and incubated at 25 °C with 12-h photoperiod for 2-3 days. Eight single-spore isolates with similar morphological characteristics were obtained and two representative isolates (P8 and S9) were characterized in depth. Colonies on PDA were initially composed of grayish-white aerial mycelium, but turned dark-gray after 7 days (Fig. 1B). Abundant black pycnidia were observed after incubation for 4 weeks. Immature conidia were hyaline, aseptate, ellipsoid, thick-walled, and mature conidia becoming dark brown and 1-septate with longitudinal striations, 25.0 - 27.0 ± 2.5 × 13.0 - 14.0 ± 1.0 µm (n = 30) (Fig.1C, D). On the basis of morphology, both representative isolates were identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (Alves et al. 2008). For molecular identification, genomic DNA of the two isolates was extracted using the DNeasy plant mini kit (Qiagen, USA). The internal transcribed spacer (ITS) region of rDNA and translation elongation factor 1-alpha (EF1-α) genes were amplified using ITS5/ITS4 and EF1-728F/EF1-986R primer set, respectively (White et al. 1990, Carbone and Kohn 1999). BLASTn analysis of the resulting ITS and EF1-α sequences indicated 100% identity to L. theobromae ex-type strain CBS 164.96 (GenBank accession nos: AY640255 and AY640258, respectively) (Phillips et al. 2013). The ITS (MW380428, MW380429) and EF1-α (MW387153, MW387154) sequences were deposited in GenBank. Phylogenetic analysis using the maximum likelihood based on the combined ITS-TEF sequences indicated that the isolates formed a strongly supported clade (100% bootstrap value) to the related L. theobromae (Kumar et al. 2016) (Fig.2). A pathogenicity test of two isolates was conducted on six healthy detached guava fruits per isolate. The fruit were surface sterilized using 70% ethanol and rinsed twice with sterile water prior inoculation. The fruit were wound-inoculated using a sterile needle according to the method of de Oliveira et al. (2014) and five-mm-diameter mycelial agar plugs from 7-days-old PDA culture of the isolates were placed onto the wounds. Six additional fruit were wound inoculated using sterile 5-mm-diameter PDA agar plugs to serve as controls. Inoculated fruit were placed in sterilized plastic container and incubated in a growth chamber at 25 ± 1 °C, 90% relative humidity with a photoperiod of 12-h. The experiment was conducted twice. Five days after inoculation, symptoms as described above developed on the inoculated sites and caused a fruit rot, while control treatment remained asymptomatic. L. theobromae was reisolated from all symptomatic tissues and confirmed by morphological characteristics and confirmed by PCR using ITS region. L. theobromae has recently been reported to cause fruit rot on rockmelon in Thailand (Suwannarach et al. 2020). To our knowledge, this is the first report of L. theobromae causing postharvest fruit rot on guava in Malaysia. The occurrence of this disease needs to be monitored as this disease can reduce the marketable yield of guava. Preventive strategies need to be developed in the field to reduce postharvest losses.

First Report of Colletotrichum siamense causing Anthracnose on White Frangipani (Plumeria alba L.) in Malaysia.

Plumeria alba L. is a flowering plant in the family Apocynaceae and widely cultivated in Malaysia as a cosmopolitan ornamental plant. In January 2020, anthracnose lesions were observed on leaves of Plumeria alba planted in Agricultural Farm, Universiti Putra Malaysia, in Selangor state, Malaysia. The disease mainly affected the leaves with symptoms occurring with approximately a 60% disease incidence. Ten symptomatic leaves were sampled from 3 different trees in the farm. Symptoms initiated as small circular necrotic spots that rapidly enlarged into black lesions with pale brown borders. Diseased tissues (5×5 mm) were surface-sterilized with 70% ethanol for 1 min, rinsed three times with sterile distilled water, dried on sterile filter papers, plated on PDA and, incubated at 25 °C with a 12-h photoperiod. A total of seven single-spore isolates with similar colony morphologies were obtained from tissue samples. After 7 days, the colonies raised the entire margin and showed white-to-gray aerial mycelium, orange conidial masses in the center and appeared dark brown at the center of the reverse view. The conidia were 1-celled, hyaline, smooth-walled, cylindrical with narrowing at the center, averaged (13-15 µm × 3 - 4 µm) (n=40) in size. Morphological characteristics of the isolates were similar to those detailed in taxonomic description of Colletotrichum sp. (Prihastuti et al. 2009). For molecular identification, genomic DNA of two representative isolates, PL3 and PL4 was extracted from fresh mycelium using DNeasy Plant Mini Kit (Qiagen, USA). The internal transcribed spacer (ITS) region, actin (ACT) and calmodulin (CAL) genes were amplified using ITS5/ITS4 (White et al. 1990), ACT-512F/783R (Carbone and Kohn 1999) and CL1C/CL2C primer sets (Weir et al. 2012). A BLAST nucleotide search of GenBank using ITS sequences showed 100% identity to Colletotrichum siamense ex-type culture ICMP 18578 (GenBank accession no. JX010171). ACT and CAL sequences showed 100% identity with C. siamense ex-type isolate BPD-I2 (GenBank accession no. FJ907423 and FJ917505). The sequences were deposited in GenBank (ITS: accession nos. MW335128, MT912574), ACT: accession nos. MW341257, MW341256, CAL: accession nos. MW341255 and MT919260). Based on these morphological and molecular characteristics, the fungus was identified as C. siamense. Pathogenicity of PL3 and PL4 isolates was verified using four healthy detached leaves of Plumeria alba. The leaves were surface-sterilized using 70% ethanol and rinsed twice with sterile water before inoculation. The leaves (three inoculation sites/leaf) were wounded by puncturing with a sterile needle through the leaf cuticle and inoculated in the wound site with 10-µl of conidial suspension (1×106 conidia/ml) from 7-days-old culture on PDA. Four leaves were used as a control and were inoculated only with 10-µl of sterile distilled water. Inoculated leaves were kept in humid chambers for 2 weeks at 25 °C with 98% relative humidity on a 12-h fluorescent light/dark period. The experiment was repeated three times. Anthracnose symptoms were observed on all inoculated leaves after 3 days, whereas controls showed no symptoms. Fungal isolates from the diseased leaves showed the same morphological characteristics as isolates PL3 and PL4, confirming Koch's postulates. C. siamense has been reported causing anthracnose on rose (Rosa chinensis) in China (Feng et al. 2019), Coffea arabica in Thailand (Prihastuti et al. 2009) and mango leaf anthracnose in Vietnam (Li et al. 2020). To our knowledge, this is the first report of Colletrotrichum siamense causing leaf anthracnose on Plumeria alba in Malaysia. Accurate identification of this pathogen provides a foundation in controlling anthracnose disease on Plumeria alba.

Comparison of anterior and posterior trabecular bone microstructure of human mandible using cone-beam CT and micro CT.

BACKGROUND: The aim of this study was to compare the trabecular bone microstructures of anterior and posterior edentulous regions of human mandible using cone-beam computed tomography (CBCT) and micro computed tomography (µCT). METHODS: Twenty volumes of interests consisting of six anterior and fourteen posterior edentulous regions were obtained from human mandibular cadavers. A CBCT system with a resolution of 80 µm (3D Accuitomo 170, J. Morita, Kyoto, Japan) and a µCT system with a resolution of 35 µm (SkyScan 1173, Kontich, Belgium) were used to scan the mandibles. Three structural parameters namely, trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were analysed using CTAn software (v 1.11, SkyScan, Kontich, Belgium). For each system, the measurements obtained from anterior and posterior regions were tested using independent sample t-test. Subsequently, all measurements between systems were tested using paired t-test. RESULTS: In CBCT, all parameters of the anterior and posterior mandible showed no significant differences (p > 0.05). However, µCT showed a significant different of Tb.Th (p = 0.023) between anterior and posterior region. Regardless of regions, the measurements obtained using both imaging systems were significantly different (p ≤ 0.021) for Tb.Th and Tb.N. CONCLUSIONS: The current study demonstrated that only the variation of Tb.Th between anterior and posterior edentulous region of mandible can be detected using µCT. In addition, CBCT is less feasible than µCT in assessing trabecular bone microstructures at both regions.

Asp-376-Glu substitution endows target-site resistance to AHAS inhibitors in Limnocharis flava, an invasive weed in tropical rice fields.

Limnocharis flava (L.) Buchenau is a problematic weed in rice fields and water canals of Southeast Asia, and in Malaysia this invasive aquatic weed species has evolved multiple resistance to synthetic auxin herbicide and acetohydroxyacid synthase (AHAS) inhibitors. In this study, it was revealed that, a single nucleotide polymorphism (SNP) at amino acid position 376, where C was substituted to G at the third base of the same codon (GAC to GAG), resulting in Aspartate (Asp) substitution by Glutamate (Glu) was the contributing resistance mechanism in the L. flava population to AHAS inhibitors. In vitro assay further proved that, all the L. flava individuals carrying AHAS resistance mutation exhibited decreased-sensitivity to AHAS inhibitors at the enzyme level. In the bensulfuron-methyl whole-plant bioassay, high resistance indices (RI) of 328- and 437-fold were recorded in the absence and presence of malathion (the P450 inhibitor), respectively. Similarly, translocation and absorption of bensulfuron-methyl in both resistant and susceptible L. flava populations showed no remarkable differences, hence eliminated the possible co-existence of non-target-site resistance mechanism in the resistant L. flava. This study has confirmed another new case of a target-site resistant weed species to AHAS-inhibitors.

Population data for 23 Y chromosome STR loci using the Powerplex® Y23 STR kit for the Kedayan population in Malaysia.

Genetic polymorphisms at 23 Y chromosome short tandem repeat (STRs) loci included in the Powerplex® Y23 PCR kit were successfully scored in 128 unrelated Kedayan individuals living in Sabah, East Malaysia. Complete haplotypes were recorded for all individuals and included 92 different types with 72 being unique to single male subjects. Three important forensic statistics were calculated from these data; haplotype diversity = 0.993, discriminating capacity = 0.719, and match probability = 0.015. The Kedayan appear to be most closely related to Malays and Filipinos in a multidimensional scaling plot and are separated from other mainland Asia populations including Thais and Hakka Han. These new data for Kedayan have been deposited in the YHRD database (accession number: YA004621). Our statistical analyses showed the reliability of Y-STR loci for geographically extended use in forensic casework and for studying human population history.

Population data of 23 Y chromosome STR loci for the five major human subpopulations of Ghana.

In this study, 268 samples for unrelated males belonging to the five major human subpopulation groups in Ghana (Akan, Ewe, Mole-Dagbon, Ga-Dangme and Guang) were genetically characterised for 23 Y chromosome short tandem repeat (STR) loci using the Powerplex® Y23 STR kit. A total of 263 complete haplotypes were recorded of which 258 were unique. The haplotype diversity, discriminating capacity and match probability for the pooled population data were 0.9998, 0.9627 and 0.0039, respectively. The pairwise genetic distance (RST) for the Ghanaian datasets and other reference populations deposited in the Y-STR Haplotype Reference Database (YHRD) were estimated and mapped using multidimensional scaling (MDS) plot. The Guang and Ewe were significantly different from the Akan, Mole-Dagbon and Ga-Dangme. However, the five Ghanaian datasets were all plotted close together with other African populations in the MDS data mapping.

First Report of Powdery Mildew caused by Podosphaera xanthii on Euphorbia tithymaloides in Malaysia.

Euphorbia tithymaloides L. (zig-zag plant) is a succulent, perennial shrub belonging to the Euphorbiaceae family and is widely cultivated in Malaysia for ornamental purposes and commercial values. In June 2019, typical symptoms of powdery mildew were observed on over 50% of the leaves of E. tithymaloides in a garden at Universiti Putra Malaysia, Serdang city of Selangor province, Malaysia. Initial symptoms included circular to irregular white powdery fungal colonies on both leaf surfaces and later covered the entire leaf surface. Severely infected leaves became necrotic, distorted and senesced. A voucher specimen Ma (PM001-Ma) was deposited in the Mycology laboratory, Faculty of Agriculture, UPM. Microscopic observation showed hyphae hyaline, branched, thin-walled, smooth, 3 to 6 µm wide with nipple-shaped appressoria. Conidiophores were straight, measured 30 to 90 µm long × 8 to 12 µm wide and composed of a cylindrical foot cell, 50 to 75 µm long. Conidia formed in chains were hyaline, ellipsoid to oval with fibrosin bodies, measured 25 to 36 × 16 to 20.1 µm in size and chasmothecia were not observed on the infected leaves. Genomic DNA was directly isolated from mycelia and conidia of isolate Ma using DNeasy Plant Mini Kit (Qiagen, USA). The universal primer pair ITS4/ITS5 of rDNA (White et al. 1990) was used for amplification and the resulting 569-bp sequence was deposited in GenBank (Accession no. MT704550). A BLAST nucleotide search revealed 100% similarity with that of Podosphaera xanthii on Momordica charantia wild from Taiwan (Accession no. KM505135) (Kirschner and Liu 2015). Both the morphological characteristics of the anamorph and ITS sequence data support the identification of this powdery mildew on E. tithymaloides as Podosphaera xanthii (Castagne) U. Braun & Shishkoff (Braun and Cook 2012). A pathogenicity test was conducted by gently pressing the infected leaves onto young leaves of five healthy potted plants. Five noninoculated plants were used as controls. The inoculated plants were maintained in a greenhouse at 25 ± 2°C and the test was repeated. Seven days after inoculation, white powdery symptoms were observed similar to those on the naturally infected leaves, while control plants remained asymptomatic. The fungus on the inoculated leaves was morphologically and molecularly identical to the fungus on the original specimens. Sequence alignments were made using MAFFT v.7.0 (Katoh et al. 2019) and a maximum likelihood phylogram was generated by MEGA v.7.0 (Kumar et al. 2016). Isolate Ma grouped in a strongly supported clade (100% bootstrap value) with the related species of P. xanthii available in GenBank based on the ITS region. Powdery mildew caused by P. xanthii has been reported as a damaging disease that can infect a broad range of plants worldwide (Farr and Rossman 2020). It also has been recently reported on Sonchus asper in China (Shi et al. 2020). According to our knowledge, this is the first report of powdery mildew caused by P. xanthii on E. tithymaloides worldwide. The occurrence of powdery mildew on E. tithymaloides could pose a serious threat to the health of this plant, resulting in death and premature senescence of young leaves.

First Report of Nigrospora sphaerica causing leaf spot on watermelon (Citrullus lanatus L.) in Malaysia.

Watermelon (Citrullus lanatus L.) is widely cultivated and consumed in Malaysia for its nutritional value. In June 2018, nearly 40% of the 'Red Rocky' watermelon plants in experimental plots of the research farm at Faculty of Agriculture, UPM Serdang, Selangor, Malaysia had leaf spot symptoms. Leaf spots were small, ranging 5 to 30 mm, yellow to brown, and circular to irregular in shape. With ages, the leafspots gradually enlarged and coalesced. To investigate the disease, ten symptomatic leaves were collected from the experimental plots. Diseased tissues (5 x 5 mm) were excied and surface sterilized with 0.5% sodium hypochlorite (NaOCl) for 2 min, rinsed twice with sterile distilled water, plated on potato dextrose agar (PDA), and incubated at 25 °C for 5 days. A total of ten isolates with similar colony morphologies were obtained from tissue samples. A single representative isolate "F" was further characterized by molecular analysis. All colonies were initially white in color, but later turned gray to black upon sporulation after 7 days. Conidia were produced in culture and were single-celled, black, smooth-walled, spherical in shape measuring 11.4 to 14 µm x 13.8 to 19 µm in diameter (n=40). These were borne on hyaline vesicles at the tip of a conidiophore. For molecular identification, genomic DNA was extracted from fresh mycelium of isolate F using DNeasy Plant Mini kit (Qiagen, Germantown, MD, USA). The internal transcribed spacer (ITS) region of rDNA and the translation elongation factor 1-alpha (TEF1-α) gene were amplified using the ITS5/ITS4 (White et al. 1990) and EF1-728F/EF1-986R primer sets (Carbone and Kohn 1999), respectively. BLASTn analysis of the ITS sequence revealed 100% identity (526 bp out of 526 bp) to Nigrospora sphaerica (GenBank Accession no. HQ608063). TEF1-α sequence had 100% identity (494 bp out 494 bp) with N. sphaerica (GenBank Accession no. MN995332). The resulting sequences were deposited in GenBank (ITS: Accession no. MK544066; TEF1-α Accession no. MT708197). Based on morphological and molecular characteristics, isolate "F" was identified as Nigrospora sphaerica (Sacc.) Mason (Chen et al. 2018). A pathogenicity test was conducted on five healthy leaves of five one-month-old watermelon 'Red Rocky' plants grown in a greenhouse. Leaves were wounded using a 34-mm-diameter florist pin frog and spray-inoculated until runoff with a conidial suspension (1 × 106 conidia/ml) of a 7-day-old culture. Five leaves from additional 2 plants were sprayed with sterile distilled water to serve as controls. Inoculated plants were covered with polyethylene bags for 48 h to maintain high humidity. Ten days post-inoculation, symptoms on inoculated leaves developed brown-to-black lesions similar to those observed in the field, while control leaves remained asymptomatic. N. sphaerica was re-isolated from all symptomatic tissues confirming Koch's postulates. N. sphaerica is distributed on a wide range of hosts and has been reported from 40 different host genera including monocotyledonous and dicotyledonous hosts (Wang et al. 2017). N. sphaerica has been reported to cause leaf spot of date palm in Pakistan (Alam et al. 2020) and kiwifruit in China (Chen et al. 2016). To our knowledge, this is the first report of N. sphaerica causing leaf spot of watermelon in Malaysia. This new disease could reduce fruit quality since sweetness and ripening are dependent on healthy foliage. Additionally, this disease can cause premature defoliation which would also reduce watermelon productivity.

First Report of Neoscytalidium dimidiatum Causing Fruit Rot on Guava (Psidium guajava L.) in Malaysia.

Guava (Psidium guajava L.) is an economically important fruit crop in Malaysia with annual production of 67,087 tons in 2018 (FAO 2018). In February 2019, fruit rot symptoms were observed postharvest on approximately 30% of guava cv. Lohan collected from a commercial orchard in the Rawang district (3°23'22.8"N 101°26'55.7"E) of Selangor province, Malaysia. Symptoms on the fruits appeared as small, circular brown spots (ranging 5 to 20 mm in diameter) that coalesced and rapidly expanded to cover the entire fruit. Severely infected fruits became soft and rotted. Ten diseased guava fruits were collected from the sampling location. Small pieces (5x5x5 mm) of symptomatic fruit tissues were excised from the lesion margin, surface-sterilized with 0.5% sodium hypochlorite (NaOCl) for 1 min, rinsed twice with sterile distilled water, plated on potato dextrose agar (PDA) and incubated at 25 °C for 5 days. A Scytalidium-like fungus was consistently isolated from symptomatic tissues on PDA after 4 days. For morphological identification, single-spore cultures were grown on PDA at 25 °C and a representative isolate LB1 was characterized further. The fungal colonies were initially white, powdery, and later turned grayish-black with the onset of sporulation. The mycelia were branched with septa, pigmented, and brown in color. Fungal colonies produced dark-brown arthroconidia with thick-walled, 0 to 1-septa, averaged 9 µm x 5 µm (n=20), and cylindrical to oblong in shape. For molecular identification, genomic DNA was extracted from fresh mycelium of isolate LB1 using DNeasy Plant Mini kit (Qiagen, Germantown, MD, USA). The internal transcribed spacer (ITS) region of rDNA and translation elongation factor 1-alpha (TEF1-α) gene were amplified using ITS5/ITS4 (White et al. 1990) and EF1-728F/EF1-986R primer set (Carbone and Kohn 1999), respectively. Both ITS (954 bp) and TEF1-α (412 bp) sequences exhibited 100% identity to Neoscytalidium dimidiatum with GenBank accession numbers FM211432 and MK495414, respectively. The resulting sequences were deposited in GenBank (ITS: Accession no. MT565490; TEF1-α Accession no. MT572846). Based on the morphological and molecular data, the pathogen was identified as N. dimidiatum (Penz.) Crous & Slippers (Crous et al. 2006). A pathogenicity test was conducted on 5 healthy detached mature guava fruits cv. Lohan by wound-inoculating using a sterile needle and pipetting 10-µl of a conidial suspension (1 × 106 conidia/ml) of isolate LB1 to the wound. Five additional fruits were wounded and pipetted 10-µl sterile distilled water to serve as controls. Inoculated fruits were placed in sterilized plastic container and incubated at 25 ± 1 °C, 90% relative humidity with a photoperiod of 12 h, and the experiment was conducted twice. All inoculated fruits developed symptoms as described above 4 to 7 days post-inoculation, while the control fruits remained asymptomatic. N. dimidiatum was re-isolated from all symptomatic tissues confirming Koch's postulates. N. dimidiatum has been reported causing brown spot disease on pitaya (Lan et al. 2012), and stem canker on dragon fruit in Malaysia and Florida (Mohd et al. 2013; Sanahuja et al. 2016) but this is the first report of N. dimidiatum causing postharvest fruit rot on guava in Malaysia. This disease can cause significant postharvest losses to guava production which could lower marketable yield and proper control strategies should be implemented.

First report of Colletotrichum siamense Causing Blossom Blight on Thai basil (Ocimum basilicum L.) in Malaysia.

Thai basil (Ocimum basilicum L.) is widely cultivated in Malaysia and commonly used for culinary purposes. In March 2019, necrotic lesions were observed on the inflorescences of Thai basil plants with a disease incidence of 60% in Organic Edible Garden Unit, Faculty of Agriculture in the Serdang district (2°59'05.5"N 101°43'59.5"E) of Selangor province, Malaysia. Symptoms appeared as sudden, extensive brown spotting on the inflorescences of Thai basil that coalesced and rapidly expanded to cover the entire inflorescences. Diseased tissues (4×4 mm) were cut from the infected lesions, surface disinfected with 0.5% NaOCl for 1 min, rinsed three times with sterile distilled water, placed onto potato dextrose agar (PDA) plates and incubated at 25°C under 12-h photoperiod for 5 days. A total of 8 single-spore isolates were obtained from all sampled inflorescence tissues. The fungal colonies appeared white, turned grayish black with age and pale yellow on the reverse side. Conidia were one-celled, hyaline, subcylindrical with rounded end and 3 to 4 µm (width) and 13 to 15 µm (length) in size. For fungal identification to species level, genomic DNA of representative isolate (isolate C) was extracted using DNeasy Plant Mini Kit (Qiagen, USA). Internal transcribed spacer (ITS) region, calmodulin (CAL), actin (ACT), and chitin synthase-1 (CHS-1) were amplified using ITS5/ITS4 (White et al. 1990), CL1C/CL2C (Weir et al. 2012), ACT-512F/783R, and CHS-79F/CHS-345R primer sets (Carbone and Kohn 1999), respectively. A BLAST nucleotide search of ITS, CHS-1, CAL and ACT sequences showed 100% similarity to Colletotrichum siamense ex-type cultures strain C1315.2 (GenBank accession nos. ITS: JX010171 and CHS-1: JX009865) and isolate BPDI2 (CAL: FJ917505, ACT: FJ907423). The ITS, CHS-1, CAL and ACT sequences were deposited in GenBank as accession numbers MT571330, MW192791, MW192792 and MW140016. Pathogenicity was confirmed by spraying a spore suspension (1×106 spores/ml) of 7-day-old culture of isolate C onto 10 healthy inflorescences on five healthy Thai basil plants. Ten infloresences from an additional five control plants were only sprayed with sterile distilled water and the inoculated plants were covered with plastic bags for 2 days and maintained in a greenhouse at 28 ± 1°C, 98% relative humidity with a photoperiod of 12-h. Blossom blight symptoms resembling those observed in the field developed after 7 days on all inoculated inflorescences, while inflorescences on control plants remained asymptomatic. The experiment was repeated twice. C. siamense was successfully re-isolated from the infected inflorescences fulfilling Koch's postulates. C. siamense has been reported causing blossom blight of Uraria in India (Srivastava et al. 2017), anthracnose on dragon fruit in India and fruits of Acca sellowiana in Brazil (Abirami et al. 2019; Fantinel et al. 2017). This pathogen can cause a serious threat to cultivation of Thai basil and there is currently no effective disease management strategy to control this disease. To our knowledge, this is the first report of blossom blight caused by C. siamense on Thai basil in Malaysia.