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

Insect pests of vegetables in Malaysia and their management using entomopathogenic fungi

The domestic economy in Malaysia is dominated by agriculture, which accounts for about 10% of the Gross Domestic Product (GDP), and contributed more than 23% of the total export earnings. Vegetable comprises about 15% of the daily food intake of the Malaysian population. Vegetables in Malaysia are mostly produced by smallholder farmers and this industry provides millions of employments for the peoples, especially the rural dwellers. Insect pest is one of the major constraints of vegetable production in Malaysia. During growth, different species of insect pests such as armyworms, caterpillars, beetles, aphids, whiteflies, mites, and thrips cause serious damage to different vegetable crops, which reduce yield and make it unsuitable for human consumption. Integrated Pest Management (IPM) has been internationally recognized approach to pest control. IPM programme for insect pest of vegetable include, cultural control, physical control, mechanical control, biological control (parasitoids, predators, and entomopathogens), Plant Resistance to insects (PRI), and chemical control. Comparably, entomopathogenic fungi has demonstrated advantageous performance in infecting hosts directly through the integument. This paper aims to review some of the available literature on the IPM of insect pests of vegetables in Malaysia with more emphasis on insect pathogenic fungi as a contribution to components of IPM

Effect of temperature on germination, radial growth, and sporulation of the new isolates of Metarhizium anisopliae and their virulence to whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae)

Metarhizium anisopliae is a potential entomopathogenic fungi (EPF) to control insect pests in Malaysia, yet little is known about the temperature influence on its growth and efficacy. The average daily temperature in Malaysia is between 21 °C and 32 °C, with some parts of Cameron Highland in Peninsular Malaysia experiencing temperatures as low as 14 °C. Therefore, for effective field application, the most effective EPF isolates should be tolerant to the temperature of the country. Here we conducted a laboratory experiment to determine the effects of average daily temperatures in Malaysia (15, 20, 25, and 30 °C) on conidial germination, radial growth, and conidial density of seven new isolates of M. anisopliae. However, at 25 and 30°C, the three isolates (Ma-2, Ma-9, and Ma-15) produced the highest number of conidia and were therefore selected for virulence test against the second instar of whitefly, Bemisia tabaci. The percentage mortality of the three isolates ranged between 36.1% and 97.7% across different temperatures for 7 days post-treatment. The isolates Ma-15 caused the highest percentage mortality of 97.7% and the shortest LT50 and LT90 values (1.68 and 3.98 days, respectively), at 30 °C incubation temperature. This study confirms that the growth and virulence of Malaysian isolates of M. anisopliae are strongly influenced by temperature and could be used as promising candidates for biocontrol of B. tabaci on vegetables planted in a tropical climate such as that of low land and highland in Malaysia, although extensive field and semi-field bioassays, as well as the development of ideal formulation, are still required

Effect of temperature on germination, radial growth, and sporulation of the new isolates of Metarhizium anisopliae and their virulence to Whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae)

Metarhizium anisopliae is a potential entomopathogenic fungi (EPF) to control insect pests in Malaysia, yet little is known about the temperature influence on its growth and efficacy. The average daily temperature in Malaysia is between 21 °C and 32 °C, with some parts of Cameron Highland in Peninsular Malaysia experiencing temperatures as low as 14 °C. Therefore, for effective field application, the most effective EPF isolates should be tolerant to the temperature of the country. Here we conducted a laboratory experiment to determine the effects of average daily temperatures in Malaysia (15, 20, 25, and 30 °C) on conidial germination, radial growth, and conidial density of seven new isolates of M. anisopliae. However, at 25 and 30°C, the three isolates (Ma-2, Ma-9, and Ma-15) produced the highest number of conidia and were therefore selected for virulence test against the second instar of whitefly, Bemisia tabaci. The percentage mortality of the three isolates ranged between 36.1% and 97.7% across different temperatures for 7 days post-treatment. The isolates Ma-15 caused the highest percentage mortality of 97.7% and the shortest LT50 and LT90 values (1.68 and 3.98 days, respectively), at 30 °C incubation temperature. This study confirms that the growth and virulence of Malaysian isolates of M. anisopliae are strongly influenced by temperature and could be used as promising candidates for biocontrol of B. tabaci on vegetables planted in a tropical climate such as that of low land and highland in Malaysia, although extensive field and semi-field bioassays, as well as the development of ideal formulation, are still required

Discovery of known and novel viruses in wild and cultivated blueberry in Florida through viral metagenomic approaches

Southern highbush blueberry (interspecific hybrids of Vaccinium corymbosum L.) is cultivated near wild V. corymbosum as well as closely related species in Florida, USA. The expansion of blueberry cultivation into new areas in Florida and deployment of new cultivars containing viruses can potentially increase the diversity of viruses in wild and cultivated V. corymbosum. In this study, viral diversity in wild and cultivated blueberries (V. corymbosum) is described using a metagenomic approach. RNA viromes from V. corymbosum plants collected from six locations (two cultivated and four wild) in North Central Florida were generated by high throughput sequencing (HTS) and analyzed using a bioinformatic analysis pipeline. De novo assembled contigs obtained from viromes of both commercial and wild sites produced sequences with similarities to plant virus species from a diverse range of families (Amalgaviridae, Caulimoviridae, Endornaviridae, Ophioviridae, Phenuiviridae, and Virgaviridae). In addition, this study has enabled the identification of blueberry latent virus (BlLV) and blueberry mosaic associated ophiovirus (BlMaV) for the first time in Florida, as well as a tentative novel tepovirus (blueberry virus T) (BlVT) in blueberry. To the best of our knowledge, this is the first study that compares viral diversity in wild and cultivated blueberry using a metagenomic approach

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 () 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

Incidence and differentiation of Cucumber mosaic virus (CMV) isolates in Peninsular Malaysia

A survey on the occurrence and incidence of Cucumber mosaic virus (CMV) was carried out on selected horticultural crops in Peninsular Malaysia. A total of 1293 leaf samples showing virus-like symptoms comprising of chilli (527 samples), tomato (63 samples), eggplant (183 samples), cucumber (209 samples), pineapple (161 samples), long bean (49 samples), and loofah (101 samples) were collected from the field at 35 different locations in 7 states (Selangor, Pahang, Melaka, Johor, Kelantan, Perak and Pulau Pinang) to detect the presence of CMV. The detection of CMV was done using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Based on the DAS-ELISA results, CMV was detected in 9% of the entire surveyed crops. The highest percentage of CMV incidences was found in Selangor with 48% infection, followed by Kelantan and Melaka (15%),Pahang (8%), Johor (7%), Pulau Pinang (4%), and Perak (1%). Amongst the samples collected, CMV was discovered to be the most prevalent on eggplant and chilli with 12% disease incidence each, followed by tomato (10%), cucumber (5%), long beans (4%) and pineapple and loofah (3% each). CMV-infected samples detected by DAS-ELISA were used in reversetranscription polymerase chain reaction (RT-PCR) analysis. Gene-specific primers were designed and a primer was then selected to amplify the region of coat protein (CP) gene, producing a specific band sized of approximately 600 base pair (bp). Samples used for RT-PCR included crops collected from Selangor, Pahang, Melaka, Johor, Kelantan, Perak, Pulau Pinang and with an addition of samples from Terengganu. Only crops collected from Johor (long beans, eggplant, cucumber, chilli and pineapple), Pahang (chilli, tomato, cucumber and eggplant), Terengganu (chilli, loofah and eggplant),Perak (cucumber), Pulau Pinang (chilli) and Selangor (chilli, tomato and eggplant) produced DNA bands of approximately 600 bp in size. Nucleotide sequences of the CP gene of 20 CMV isolates from tomato,eggplant, loofah, chilli and tobacco collected in Terengganu, Pahang,Penang, Perak and Selangor were confirmed as CMV. Phylogenetic analysis of the partial CP genes suggest that the local CMV isolates belong to subgroup I (S-I) and are grouped together with the subgroup IB (S-IB) strains. The results further proposed that the isolates can be arranged according to the type of crops and geographical location

Known and New Emerging Viruses Infecting Blueberry

Blueberry (Vaccinium spp.) plants are exposed to existing and emerging viruses as a result of expanding acreage of blueberry plantations across the world, primarily in North America. Since blueberry is cultivated in areas where there are wild Vaccinium spp., there is increasing risk of virus movement between wild and cultivated blueberries. This is theoretically possible because viruses can spread from commercial cultivars to native species and vice versa causing the spread of existing and new viruses. The occurrence of these viruses in blueberry can be devastating to the industry considering the cost for cultivation and production of this perennial crop. However, the advent of high-throughput sequencing and bioinformatic sequence analysis have allowed for rapid identification of known and novel viruses in any crop including blueberry, thus facilitating proper intervention in response to serious viral diseases. In this paper, we aim to focus on the current status of known and novel viruses emerging in blueberry worldwide, which may impact the blueberry industry

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

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