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.

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.

Mycotoxigenic fungal growth inhibition and multi-mycotoxin reduction of potential biological control agents indigenous to grain maize.

The present work investigated the potential of fungal species from grain maize farms in Malaysia as antagonists against the indigenous mycotoxigenic fungal species and their subsequent mycotoxin production. Dual-culture assay was conducted on grain maize agar (GMA) with 12 strains of potential fungal antagonists namely Bjerkandra adusta, Penicillium janthinellum, Schizophyllum commune, Trametes cubensis, Trichoderma asperelloides, Trichoderma asperellum, Trichoderma harzianum, and Trichoderma yunnanense against seven mycotoxigenic strains namely Aspergillus flavus, Aspergillus niger, Fusarium verticillioides, and Fusarium proliferatum producing aflatoxins, ochratoxin A, and fumonisins, respectively. Based on fungal growth inhibition, Trichoderma spp. showed the highest inhibitory activity (73-100% PIRG, Percentage Inhibition of Radial Growth; 28/0 ID, Index of Dominance) against the tested mycotoxigenic strains. Besides, B. adusta and Tra. cubensis showed inhibitory activity against some of the tested mycotoxigenic strains. All fungal antagonists showed varying degrees of mycotoxin reduction. Aflatoxin B1 produced by A. flavus was mainly reduced by P. janthinellum, Tra. cubensis, and B. adusta to 0 ng/g. Ochratoxin A produced by A. niger was mainly reduced by Tri. harzianum and Tri. asperellum to 0 ng/g. Fumonisin B1 and FB2 produced by F. verticillioides was mainly reduced by Tri. harzianum, Tri. asperelloides, and Tri. asperellum to 59.4 and 0 µg/g, respectively. Fumonisin B1 and FB2 produced by F. proliferatum were mainly reduced by Tri. asperelloides and Tri. harzianum to 244.2 and 0 µg/g, respectively. This is the first study that reports on the efficacy of Tri. asperelloides against FB1, FB2, and OTA, P. janthinellum against AFB1, and Tra. cubensis against AFB1.

Inoculation and colonization of the entomopathogenic fungi, Isaria javanica and Purpureocillium lilacinum, in tomato plants, and their effect on seedling growth, mortality and adult emergence of Bemisia tabaci (Gennadius).

Entomopathogenic fungi (EPF) are natural enemies which affect insect population and have long been recognized as biological control agents against many insect pests. Some isolates have also been established as endophytes, benefiting their host plants without causing any symptoms or negative effects. Here we demonstrated two entomopathogenic fungal species, Isariajavanica (Frieder. & Bally) Samson & Hywel-jone 2005 and Purpureocillium lilacinum (Thom) Luangsa-ard, Hou-braken, Hywel-Jones & Samson (2011) as endophytes in tomato plants by using the seed inoculation method and examined their effect on plant growth, B. tabaci mortality, and adult emergence. Our study indicated that tomato seeds treated with a fungal suspension of I. javanica and P. lilacinum enabled their recovery from plant tissues (root, stem and leaf) up to 60 days after inoculation (DAI). Both endophytic isolates also caused significant mortality of adult B. tabaci on seedlings inoculated with, I. javanica (51.92±4.78%), and P. lilacinum (45.32±0.20%) compared to the control treatment (19.29±2.35). Adult emergence rates were significantly high in the control treatments (57.50±2.66%) compared to I. javanica (15.00±1.47%) and P. lilacinum (28.75±4.78%) treatments. This study provides evidence that endophytic isolates of I. javanica and P. lilacinum have a biocontrol potentials for used against whiteflies and could also explored as plant growth promoters.

Streptomyces-mediated growth enhancement and Bacterial Panicle Blight disease suppression in rice plants under greenhouse conditions.

Streptomyces corchorusii TKR8, Streptomyces corchorusii JAS2 and Streptomyces misionensis TBS5 were previously obtained from rice fields and have been studied as a biocontrol agent against the causal agent of Bacterial Panicle Blight (BPB) disease on rice, Burkholderia glumae, and rice plant growth promoter. This study evaluated the potential of plant growth-promoting Streptomyces (PGPS) to control B. glumae and promote rice plants' growth under greenhouse conditions. PGPS were further characterized based on their phenotypic and biochemical differences. Multilocus sequence analysis (MLSA) by amplifying gyrB, rpoB and trpB using PCR was conducted to identify the PGPS further. The antimicrobial activity of PGPS against B. glumae was investigated using a survival assay and microscopic analysis. Result indicates that JAS2 (61.2 %) utilized the highest number of carbohydrates tested, followed by TKR8 (53.1 %) and TBS5 (40.8 %) as analyzed using API 50 CH. Based on MLSA analysis of the concatenated partial sequences (1520 bp) from three housekeeping genes, the neighbor-joining tree identified JAS2 and TKR8 as S. corchorusii. Meanwhile, TBS5 as S. misionensis. Antimicrobial activity of PGPS against B. glumae has found that the supernatant of Streptomyces reduced the survival viability of B. glumae up to 50.7-70.3 %. SEM images showed that substantial morphological changes happened in cell membranes of B. glumae after the Streptomyces treatment. The highest vigor index of inoculated seedlings was determined when rice seeds were treated with a spore suspension of 1 × 107 spore/mL (for JAS2 and TKR8) and 1 × 106 spore/mL (for TBS5). Under greenhouse conditions, Streptomyces-treated plants showed improvement in rice plants' growth and grain yield and reduced the BPB disease severity. Results suggest that the S. corchorusii TKR8, S. corchorusii JAS2 and S. misionensis TBS5 should be promoted as biocontrol agents against B. glumae and bioformulations for rice crops.

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.

Current Classification of the Bacillus pumilus Group Species, the Rubber-Pathogenic Bacteria Causing Trunk Bulges Disease in Malaysia as Assessed by MLSA and Multi rep-PCR Approaches.

Bacillus pumilus is the causal agent of trunk bulges disease affecting rubber and rubberwood quality and yield production. In this study, B. pumilus and other closely related species were included in B. pumilus group, as they shared over 99.5% similarity from 16S rRNA analysis. Multilocus sequence analysis (MLSA) of five housekeeping genes and repetitive elements-based polymerase chain reaction (rep-PCR) using REP, ERIC, and BOX primers conducted to analyze the diversity and systematic relationships of 20 isolates of B. pumilus group from four rubber tree plantations in Peninsular Malaysia (Serdang, Tanah Merah, Baling, and Rawang). Multi rep-PCR results revealed the genetic profiling among the B. pumilus group isolates, while MLSA results showed 98-100% similarity across the 20 isolates of B. pumilus group species. These 20 isolates, formerly established as B. pumilus, were found not to be grouped with B. pumilus. However, being distributed within distinctive groups of the B. pumilus group comprising of two clusters, A and B. Cluster A contained of 17 isolates close to B. altitudinis, whereas Cluster B consisted of three isolates attributed to B. safensis. This is the first MLSA and rep-PCR study on B. pumilus group, which provides an in-depth understanding of the diversity of these rubber-pathogenic isolates in Malaysia.

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.

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.

Plant Growth-Promoting Bacteria as an Emerging Tool to Manage Bacterial Rice Pathogens.

As a major food crop, rice (Oryza sativa) is produced and consumed by nearly 90% of the population in Asia with less than 9% produced outside Asia. Hence, reports on large scale grain losses were alarming and resulted in a heightened awareness on the importance of rice plants' health and increased interest against phytopathogens in rice. To serve this interest, this review will provide a summary on bacterial rice pathogens, which can potentially be controlled by plant growth-promoting bacteria (PGPB). Additionally, this review highlights PGPB-mediated functional traits, including biocontrol of bacterial rice pathogens and enhancement of rice plant's growth. Currently, a plethora of recent studies address the use of PGPB to combat bacterial rice pathogens in an attempt to replace existing methods of chemical fertilizers and pesticides that often lead to environmental pollutions. As a tool to combat bacterial rice pathogens, PGPB presented itself as a promising alternative in improving rice plants' health and simultaneously controlling bacterial rice pathogens in vitro and in the field/greenhouse studies. PGPB, such as Bacillus, Pseudomonas, Enterobacter, Streptomyces, are now very well-known. Applications of PGPB as bioformulations are found to be effective in improving rice productivity and provide an eco-friendly alternative to agroecosystems.

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.

Phytopathogenic organisms and mycotoxigenic fungi: Why do we control one and neglect the other? A biological control perspective in Malaysia.

In this review, we present the current information on development and applications of biological control against phytopathogenic organisms as well as mycotoxigenic fungi in Malaysia as part of the integrated pest management (IPM) programs in a collective effort to achieve food security. Although the biological control of phytopathogenic organisms of economically important crops is well established and widely practiced in Malaysia with considerable success, the same cannot be said for mycotoxigenic fungi. This is surprising because the year round hot and humid Malaysian tropical climate is very conducive for the colonization of mycotoxigenic fungi and the potential contamination with mycotoxins. This suggests that less focus has been made on the control of mycotoxigenic species in the genera Aspergillus, Fusarium, and Penicillium in Malaysia, despite the food security and health implications of exposure to the mycotoxins produced by these species. At present, there is limited research in Malaysia related to biological control of the key mycotoxins, especially aflatoxins, Fusarium-related mycotoxins, and ochratoxin A, in key food and feed chains. The expected threats of climate change, its impacts on both plant physiology and the proliferation of mycotoxigenic fungi, and the contamination of food and feed commodities with mycotoxins, including the discovery of masked mycotoxins, will pose significant new global challenges that will impact on mycotoxin management strategies in food and feed crops worldwide. Future research, especially in Malaysia, should urgently focus on these challenges to develop IPM strategies that include biological control for minimizing mycotoxins in economically important food and feed chains for the benefit of ensuring food safety and food security under climate change scenarios.

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 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.

Cytotoxicity and Anticancer Activity of Donkioporiella mellea on MRC5 (Normal Human Lung) and A549 (Human Lung Carcinoma) Cells Lines.

Polypores are mushrooms which are rich in bioactivities and for generations, they have been widely used as herbal remedies. Despite their significant importance in treatments of various health issues, only a few local species have been reported for their pharmacological potentials. The present study was carried out to establish cytotoxicity potentials of Donkioporiella mellea, a local polypore species collected from forested areas in Malaysia at cellular levels on normal human lung (MRC5) and human lung carcinoma (A549) cell lines. Survival and inhibition rates were analyzed by 3-(4, 5)-dimethylthiahiazo (-z-y-l)-2,5-diphenyltetrazoliumbromide (MTT) while monitoring changes on cellular shapes by inverted phase contrast microscopy. Survival rates of MRC5 cells were observed to be significantly higher than A549 after treatments with various concentrations of polypore extracts. MRC5 cells showed excellence in survival performance when treated with hot and cold aqueous extracts. Cold aqueous extract showed higher cytotoxicity activities compared to hot aqueous extract (p < 0.0001) with inhibitory concentration (IC50) values of 414.29 µg/ml and >1000 µg/ml, respectively. Treatments with tamoxifen as a control exhibited necrotic features in both cell lines. The results suggest that D. mellea possesses pharmacological potentials that can be utilized for human consumption as a new bioresource alternative, thus encouraging research advancement in mycological and nutraceutical studies.

First Report of Fusarium incarnatum-equiseti Species Complex Causing Leaf Spot on Rockmelon (Cucumis melo L.) in Malaysia.

Rockmelon, (Cucumis melo L.) is an economically important crop cultivated in Malaysia. In October 2019, severe leaf spot symptoms with a disease incidence of 40% were observed on the leaves of rockmelon cv. Golden Champion at Faculty of Agriculture, Universiti Putra Malaysia (UPM). Symptoms appeared as brown necrotic spots, 10 to 30 mm in diameter, with spots surrounded by chlorotic halos. Pieces (5 x 5 mm) of diseased tissue were sterilized with 0.5% NaOCl for 1 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA) and incubated at 25°C for 7 days with a 12-h photoperiod. Nine morphologically similar isolates were obtained by using single spore isolation technique and a representative isolate B was characterized further. Colonies were abundant, whitish aerial mycelium with orange pigmentation. The isolates produced macroconidia with 5 to 6 septa, a tapered with pronounced dorsiventral curvature and measured 25 to 30 µm long x 3 to 5 µm wide. Microconidia produced after 12 days of incubation were single-celled, hyaline, ovoid, nonseptate, and 1.0 to 3.0 × 4.0 to 10.0 µm. Morphological characteristics of the isolates were similar to the taxonomic description of Fusarium equiseti (Leslie and Summerell 2006). Genomic DNA was extracted from fresh mycelium using DNeasy Plant Mini kit (Qiagen, USA). To confirm the identity of the fungus, two sets of primers, ITS4/ITS5 (White et al. 1990) and TEF1-α, EF1-728F/EF1-986R (Carbone and Kohn 1999) were used to amplify complete internal transcribed spacer (ITS) and partial translation elongation factor 1-alpha (TEF1-α) genes, respectively. BLASTn search in the NCBI database using ITS and TEF-1α sequences revealed 99 to 100% similarities with species of both F. incarnatum and F. equiseti. BLAST analysis of these in FUSARIUM-ID database showed 100% and 99% similarity with Fusarium incarnatum-F. equiseti species complex (FIESC) (NRRL34059 [EF-1α] and NRRL43619 [ITS]) respectively (Geiser et al. 2004). The ITS and TEF1-α sequences were deposited in GenBank (MT515832 and MT550682). The isolate was identified as F. equiseti, which belongs to the FIESC based on morphological and molecular characteristics. Pathogenicity was conducted on five healthy leaves of 1-month-old rockmelon cv. Golden Champion grown in 5 plastic pots filled with sterile peat moss. The leaves were surface-sterilized with 70% ethanol and rinsed twice with sterile-distilled water. Then, the leaves were wounded using 34-mm-diameter florist pin frog and inoculated by pipetting 20-µl conidial suspension (1 × 106 conidia/ml) of 7-day-old culture of isolate B onto the wound sites. Control leaves were inoculated with sterile-distilled water only. The inoculated plants were covered with plastic bags for 5 days and maintained in a greenhouse at 25 °C, 90% relative humidity with a photoperiod of 12-h. After 7 days, inoculated leaves developed necrotic lesions similar to the symptoms observed in the field while the control treatment remained asymptomatic. The fungus was reisolated from the infected leaves and was morphologically identical to the original isolate. F. equiseti was previously reported causing fruit rot of watermelon in Georgia (Li and Ji 2015) and China (Li et al. 2018). This pathogen could cause serious damage to established rockmelon as it can spread rapidly in the field. To our knowledge, this is the first report of a member of the Fusarium incarnatum-F.equiseti species complex causing leaf spot on Cucumis melo in Malaysia.

Recovery of Recurrent Parent Genome in a Marker-Assisted Backcrossing Against Rice Blast and Blight Infections Using Functional Markers and SSRs.

The most vital aspect of marker-assisted backcross breeding is the recurrent parent genome recovery. This enables the selection of only parents with recovered recipient/recurrent parent genome in addition to the targeted genes. The recurrent parent genome recovery (RPGR) ensures that non-desirable genomic segments are removed while the gene of interest is sustained in the recombined progenies without further segregations. This work was aimed at quantifying the RPGR of backcross populations with introgression of bacterial leaf blight resistance genes. Putra-1, a Malaysian elite variety, high yielding with inherent resistance to blast but susceptible to bacterial leaf blight (BLB), was crossed with IRBB60 which is resistant to BLB disease. The IRBB60 has four Xoo resistance genes-Xa4, xa5, xa13 and Xa21. Tightly linked polymorphic functional and SSR markers were used for foreground selection at every stage of backcrossing to select progenies with introgressed target genes. Background selection was done to quantify the percentage of RPGR in the selected lines using 79 confirmed polymorphic microsatellites. Result obtained showed that the percentage of RPGR was 80.11% at BC1F1, 95.30% at BC2F1 and 95.9% at BC2F2. The introgression of Xa4, xa5, xa13 and Xa21 Xoo resistance genes were faster through the adopted marker-assisted backcross breeding compared to what could be obtained through conventional breeding. All the 16 selected lines displayed resistance to BLB with three lines showing high resistance to the disease. The blast resistance contained in the genetic background of Putra-1 was also sustained in all the selected lines. The newly developed lines were recommended as new rice varieties for commercial cultivation.

A Review of the Biology and Control of Whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), with Special Reference to Biological Control Using Entomopathogenic Fungi.

Whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), consists of genetically diverse species known to cause significant destruction in several crops around the world. Nymphs and adults of B. tabaci cause damage to plants during feeding, and they can act as a virus vector, thus causing significant yield loss to crops in the tropical and subtropical regions. Chemical pesticides are widely used to control B. tabaci due to their immediate action, but this approach has several drawbacks including food safety issues, insecticide resistance, environmental pollution, and the effect on non-target organisms. A biological control agent using entomopathogenic fungi (EPF) has therefore been developed as an alternative against the conventional use of chemical pesticides in an integrated pest management (IPM) system to effectively control B. tabaci. It is apparent from this review that species of hyphomycetes fungi are the most common EPF used to effectively control B. tabaci, with the second instar being the most susceptible stage of infection. Therefore, this review article focuses specifically on the control of B. tabaci with special emphasis on the use of EPF as biological control agents and their integration in IPM.

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.