Styphnolobium japonicum (L.) Schott (family Fabaceae Juss.) also called pagoda tree, is widely planted in northern China in landscape plantings, for erosion control and forestry. In recent years, symptoms of branch dieback were observed on S. japonicum in the southern part of Xinjiang province, China. From 2019 to 2022, in total ca. 1000 ha area was surveyed in Korla (41.68°N, 86.06°E), Bohu (41.95°N, 86.53°E) and Alaer (41.15°N, 80.29°E). Typical symptoms were observed in 70% of the surveyed branches. To identify the cause, we collected 50 symptomatic branches. Symptoms were initially observed on green current-year twigs, which turned grayish white in color. In the later stages of disease development, a large number of nacked black conidia formed under epidermis of perennial branches, causing visible black protrusions (pycnidia) on branch surface. The disease occurred throughout the entire growing season of S. japonicum. Symptoms also occurred on the inflorescence, fruit, and twigs. In some cases, infection resulted in tree mortality. Isolations were made from the margin between healthy and diseased tissues. Small pieces were excised, surface disinfested (75% ethanol 30 s, 1% NaClO solution 5 mins), cut into pieces (5 to 10 mm2), and incubated on PDA medium at 28℃ for 3 days. A total of 16 isolates (GH01-GH16) with similar colony morphology were obtained. The colonies were initially white, gradually turning to olive-green on the surface and black on the underside after 7 days. Microscopically, the conidia were aseptate, 1-septate, two-septate, and muriform, 2.6-4.5 × 2.9-27.6 μm (n=50). Pycnidia ranged in size from 120.2 to 135.5 × 112.4 to 118.6 µm (n=20). Those morphological characters matched the descriptions of Neoscytalidium dimidiatum (previously N. novaehollandiae) (Alizadeh et al. 2022; Pavlic et al. 2008). For molecular identification, genomic DNA of GH01-GH16 were extracted from fresh mycelia. The internal transcribed spacer (ITS), large subunit ribosomal RNA gene (LSU), and translation elongation factor 1-alpha (EF1-α) gene were amplified using the primer sets ITS1/ITS4 (White 1990), LRoR/LR5 (Vilgalys and Hester 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999). The sequences were deposited in GenBank (accession No. OP379832, OQ096643-OQ096657 for ITS, OP389048, OQ127403-OQ127417 for LSU, and OQ136617, OQ586044-OQ586058 for EF1-α). The ITS sequence had 100% identity (505/505 bp) to MT362600. Similarly, the LSU and EF1-α sequences were found to be identical to MW883823 (100%, 821/821 bp) and KX464763(99%, 256/258 bp), respectively. Pathogenicity was tested on one-year-old healthy S. japonicum seedlings. Spores of representative isolate GH01 were produced on PDA by incubating for 7-days at 28℃. Conidia were washed with sterile water. Five trees were inoculated with 1 × 106 conidia/ml conidial suspensions and five trees were sprayed with sterile water. All trees were covered with plastic bags for 24 h and kept at 25°C in a greenhouse. Signs and symptoms were similar to those observed in field collections one month after inoculation, while no symptoms occurred on the controls. The original fungus was successfully reisolated from the inoculated trees and was identified as N. dimidiatum following the methods described above. N. dimidiatum has been reported in many Asian country such as Malaysia, India, Turkey, and Iran(Akgül et al. 2019; Alizadeh et al. 2022; Khoo et al. 2023; Salunkhe et al. 2023). To our knowledge, this is the first report of N. dimidiatum associated with branch dieback of S. japonicum in China. Our findings have expanded the host range of N. dimidiatum in China and provides a theoretical basis for the diagnosis and treatment of the disease.
Roselle (Hibiscus sabdariffa L.) is a crop of economic importance, refreshing drinks are prepared from its calyces, it is also attributed to antioxidant, antibacterial, and antihypertensive properties (Da-Costa-Rocha et al. 2014). In November 2022, in municipality of Iguala (18.355592N, 99.548546W, 749 m above sea level), Guerrero, México, roselle plants of approximately 1.5 months of age with basal rot were detected under greenhouse conditions. The symptoms consisted of wilting, yellowing, and root and stem rot with constriction in the base of the stem. The symptoms were detected in approximately 15% of plants at the operation. From symptomatic tissue, cuts were made into approximately 0.5 cm pieces, sterilized with 2% NaClO, washed with sterile distilled water, transferred to PDA medium amended with 50 mg/liter of Chloramphenicol, and incubated in the dark for four days at 28 °C. Rhizoctonia-like colonies were consistently obtained, and nine isolates were selected and purified by the hyphal-tip method. After four days, isolates developed a mycelium was light-white that became brown with age. Right-angled hyphal branching was also observed, in addition to a slight constriction at the base of the branches. In some older cultures, numerous dark brown sclerotia were observed. They were multinucleate cell with three to eight nuclei and measured from 1 to 2 mm in diameter. Together these characteristics were consistent with the description of Rhizoctonia solani Kühn (Parmeter 1970). The anastomosis group (AG) was confirmed by amplifying the ITS region with the primers ITS1 and ITS4 (White et al. 1990) of the RIJAM3 and RIJAM5 strains. The sequences were deposited in GenBank (Nos. OR364496 and OR364497 for RIJAM3 and RIJAM5, respectively). BLAST analysis, both isolates indicated 99.7 identity to R. solani AG-4 HG-I (GenBank: KM013470) strain ICMP 20043 (Ireland et al. 2015). The phylogenetic analysis of AGs sequences allowed assignment of isolates RIJAM3 and RIJAM5 to the AG-4 HG-1 clade. A pathogenicity test was performed on 20 one-month-old roselle plants. Mycelium of RIJAM3 isolate was inserted into the base of the stem with a sterile toothpick. As a control, a sterile toothpick with no mycelium was inserted in ten healthy plants. Additionally, 50 eight-day-old seedlings were inoculated by placing a 5-mm diameter agar plug colonized with mycelium of RIJAM3 at the base of the stem 10 mm below the soil surface. As control treatments, uncolonized PDA plugs were deposited at the base of 25 seedlings. The inoculated plants were incubated in a greenhouse with an average temperature and relative humidity of 28°C and 85%, respectively. Following inoculation, symptoms similar to those observed in the original outbreak were observed in plants after six days and only after four days in seedlings. In both experiments, the control plants and seedlings remained asymptomatic. R. solani was re-isolated from plants and seedlings, complying with Koch's postulates. The pathogenicity testing was repeated twice, with concordant results. In Nigeria and Malaysia R. solani was reported to seedling death to cause seedling dieback in roselle (Adeniji 1970; Eslaminejad and Zakaria 2011). In México R. solani AG-4 has been previously reported in crops of potato, chili and tomato (Montero-Tavera et al. 2013; Ortega-Acosta et al. 2022; Virgen-Calleros et al. 2000). To the best of our knowledge, this is the first report of R. solani AG-4 HG-I as a causing of root and basal stem rot on roselle in Mexico. This research provides information essential for informing the management of this disease, and may help design measures to prevent the spread of the pathogen to other regions.
Hibiscus latent Singapore virus (HLSV) and Hibiscus latent Fort Pierce virus (HLFPV) both belong to the genus Tobamovirus in the family Virgaviridae. The genomes of both HLSV and HLFPV consist of a linear positive sense single-stranded RNA of about 6.3 kb. HLSV is the causal agent of hibiscus leaf crinkle disease. Infections of HLSV in hibiscus (Hibiscus rosa-sinensis) have so far only been reported in Singapore, Japan and Malaysia (Srinivasan et al., 2002; Yoshida et al., 2018; Yusop et al., 2021). In 2017, leaf curling and chlorosis symptoms of lantana (Lantana camara) plants were found in Chenshan Botanical Garden, Shanghai, China. To detect potential virus(es) in these lantana samples, leaves from one lantana plant were collected and total RNA was extracted with RNAiso Plus (TaKaRa). A cDNA library was prepared by TruSeq RNA Sample Prep Kit (Illumina) after removing ribosomal RNA by Ribo-ZeroTM rRNA Removal Kit (Epicentre). The paired-end sequencing was then performed on an Illumina NovaSeq 6000. A total of 61,085,018 high quality reads were obtained and de novo assembly by StringTie revealed 124,516 contigs (greater than 50 bp, N50=719 bp) with an average length of 537 bp. BLASTx analyses in the National Center for Biotechnology Information (NCBI) database showed that 1 long contig of 6,305 bp, assembled of 1794 clean reads, shared significant nucleotide similarities with the genomic sequence of HLSV, and 1 contig of 6,271 bp, assembled of 3174 clean reads, shared significant similarities with the genomic sequence of HLFPV, yielding an average coverage of the whole genome at 42.65 and 75.83 per million reads, respectively. To obtain the complete genome of the viral RNA in this lantana sample, eleven overlapping regions covering the entire HLSV viral genome, and nine overlapping regions covering the entire HLFPV viral genome were amplified by reverse transcription-PCR (RT-PCR) and sequenced. In addition, the exact 5' and 3' ends of the genomic RNA of each virus were determined by rapid amplification of the cDNA ends (RACE) (Wang et al. 2020). The complete genome of the identified HLSV, deposited in GenBank: MZ020960, is 6,486 nt in length and shows 98.4% nucleotide sequence identity with HLSV Singapore isolate (GenBank: AF395898). Similar to other HLSV isolates, this virus isolate possesses an internal poly(A) tract of 87 nucleotides, which is crucial to virus replication (Niu et al., 2015). The complete genome of the Lantana HLFPV isolate is 6,463 nt (GenBank MZ020961) including a 73 nt internal poly(A) tract, and has 98.4% nt identity to HLFPV-Japan (AB917427). In two other lantana plants from the same site, the presence of HLSV and HLFPV was confirmed by RT-PCR using the primer pairs (5'-GCATCTGCATAACACGGTTG-3'/5'-ACGTTGTAGTAGACGTTGTTGTAG-3' and 5'-GGACCTTGCTAATCCGCTAAAGTTG-3'/5'-GGTCCATGTCCATCCAGATGCAATC-3'). In addition to the HLSV and HLFPV genomes, BLASTx analysis of three contigs of 3,006 bp, 2,845 bp and 2,200 bp, assembled of 1328, 352 and 2280 clean reads respectively, showed high identity to RNAs 1 (MG182148), 2 (DQ412731) and 3 (KY794710) of cucumber mosaic virus. To the best of our knowledge, this is the first report of L. camara as a new natural host of HLSV and HLFPV, and first identification of a mixed infection of HLSV and HLFPV.
In June 2017, severe leaf spots symptoms were observed by growers on pineapple leaves of Josapine variety in in Alor Pongsu (5°01'60.00" N, 100°34' 59.99" E), Perak, northwest of Peninsular Malaysia. The early infection stage shows that several brown spots could be observed, which then would merge to form large brown to creamy white lesions that cover all the leaf surface. This infection finally caused the plant to die after a while. Disease observations conducted from 2018 - 2023 showed that 10-15% incidences of the disease were observed in several pineapple farms located in Johor, Kedah, and Sarawak. The aim of this study to confirm the causal pathogen of the disease by performing isolation, pathogenicity testing, and identification of the primary causal pathogen from 20 samples of infected leaves collected from Alor Pongsu. The leaf tissues between infected and healthy were cut into small pieces (0.5 cm 0.5 cm), and surface sterilized with 1% sodium hypochlorite for 30 seconds, followed by 70% ethanol for 30 seconds, and rinsed thrice with sterilized water before placing on Potato Dextrose Agar (PDA). The PDA plates were incubated at room temperature (28 ± 2℃) in natural light. After five days of incubation, the potential causal pathogen was purified using a single conidial isolation technique for morphological and molecular characterizations. All 32 isolates displayed similar phenotypes. Based on morphological observation on PDA, the colonies were initially white of aerial mycelia but gradually darkened as the culture aged. Microscopic features of the 14-day-old fungal culture showed that the mycelia were branched with 0- 1 septa, pigmented, and brown. Arthroconidia were ellipsoid to ovoid or round shaped, hyaline, with rounded apex, truncate base, and occurring singly or in chains averaging 9 ± 3 × 5 ± 2 μm (n = 20). Based on the morphological characteristics, the fungal isolates were tentatively identified as Neoscytalidium species. A representative isolate of Neoscytalidium coded as UiTMPMD2 was further identified through PCR implication of the internal transcribed spacer (ITS) region using ITS1 and ITS4 primers and BLAST homology search as Neoscytalidium dimidiatum (Penz.) Crous & Slippers based on 100% similarity (575 bp out of 575 bp) to a reference sequence (accession no. KU204558.1). The sequence was deposited in Gen Bank (accession no. OR366479) with reference sequence code of INBio:30A. Pathogenicity tests were performed on 10 whole plants of Josapine pineapple (4 months old) using a leaf inoculating method (Wu et al. 2022) in a glasshouse (25-32°C) and repeated twice. Four mature leaves per each plant were wounded at two points and inoculated with mycelium PDA plugs from 7-days-old cultures of N. dimidiatum. Control plants were wounded in the same manner but inoculated with sterilized PDA plugs. Seven days post inoculation, leaf spot symptoms were observed on treated plants with the pathogen, while the control plants remained symptomless. Pathogen was successfully reisolated from brown leaf spot symptoms in which the cultural and morphological characteristics were identical to those of the originals. Neoscytalidium dimidiatum has a wide range of hosts and it has been reported in Malaysia to cause stem canker on pitahaya (Mohd et al. 2003; Khoo et al. 2023 ) and fruit rot of guava (Ismail et al. 2021). To the best of our knowledge this is the first report of N. dimidiatum causing leaf spots on pineapples in Malaysia. This report establishes a foundation for further study of N. dimidiatum that can effectively address the disease in pineapple.
Dragon fruit (Hylocereus polyrhizus) is a high value newly introduced fruit crop in Bangladesh. It has drawn considerable public attention due to its appealing flesh color, sweet taste and fruit qualities. Recently, basal rot of dragon fruit plants was observed in several farmer's fields, nurseries and in the research field of Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU) where about 10-15% of plants were infected in each location. Initially, the symptoms appeared in the basal part near the soil as brown lesions which gradually extended to the upper stem and finally becoming soft and watery (Figure 1a). Infected plants were collected from Kapasia of Gazipur district (Latitude 24.266 and Longitude 90.633) to isolate the causal organism. Isolations were carried out following the procedure reported by Briste et al. (2019). Briefly, infected plant parts were surface sterilized in 2% NaOCl for 1 min followed by 70% ethanol for 5 min and rinsed 3 times with sterile double distilled water. A large piece of a surface sterilized plant was cut into small pieces (2 mm × 2 mm) from the margin of the necrotic lesion and placed on half strength potato dextrose agar (PDA) and incubated for 7 days at 25 °C. The BTFD1 and BTFD4 isolates were purified from single spores resulting in white colonies with a growth rate of 1cm/day on PDA (Figure 1b). Colonies produced single celled microconidia from unbranched, short monophialidic conidiophores and septate macroconidia as well as chlamydospores in PDA which is consistent with Fusarium oxysporum (Figure 1c). To confirm the identity of the isolates, the internal transcribed spacer (ITS1, 5.8S rRNA and ITS2) and translation elongation factor-1alpha (EF-1α) were amplified using primers ITS-1/ ITS-4 and EF1-728F/ EF1-986R, respectively (Surovy et al. 2018). The ITS sequences of the isolates BTFD1 and BTFD4 (GenBank accession # MN727096 and MN727095, respectively) showed 100% similarity with the sequence from F. oxysporum strain JJF2 (MN626452). Sequence identity for EF-1α (GenBank accession # MN752123 and MN752124, respectively) was 100% with the sequence from F. oxysporum strain CAV041_EO (MK783088). The isolates (BTFD1 and BTFD4) were identified as F. oxysporum based on the aligned sequences of ITS and EF-1α, molecular phylogenetic analyses by maximum likelihood tree (Figure 2a) and maximum parsimony tree methods (Figure 2b). The isolates were stored at 4°C on dried filter paper as well as in an ultra-low temperature freezer (-80°C) at IBGE, BSMRAU, Bangladesh and are available on request. To ensure pathogenicity, isolate BTFD1 was grown on PDA, incubated at 25°C for 7 days and 250 ml conidial suspension (with 1 × 105 conidia/ml) was prepared. Twelve,three-month-old healthy dragon fruit plants were inoculated. Pathogenicity tests were carried out in two sets using three replications in each set. In one set, only the basal part of the plants was dipped into the conidial suspension and in another set the whole plant was dipped into the conidial suspension for two hours. Sterile distilled water was also used in another set of plants as a control. The inoculated plants were placed on wet tissue in a plastic box (31cm × 24cm × 8cm) covered and incubated at 25°C. After 10 days, all inoculated plants in both sets developed rot symptoms similar to those observed in the field, while the control plants remained healthy (Figure 1d). The pathogen was successfully re-isolated from the inoculated symptomatic parts on half strength PDA medium and had morphology as characterized before, thus fulfilling Koch's postulates. This disease has been reported in Argentina and Malaysia (Wright et al. 2007; Hafifi et al. 2019). To the bet of our knowledge, this is the first report of Fusarium basal rot of dragon fruit in Bangladesh caused by F. oxysporum.
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.
Crinum asiaticum (family Amaryllidaceae), locally known as 'Pokok Bakung', is an ornamental medicinal plant grown in Malaysia. It contains chemical compounds used for antimicrobial, antioxidant, antitumor, antiemetic and wound healing (Patel, 2017). In July 2021, 'Pokok Bakung' leaves with anthracnose symptoms were collected from a park of Universiti Malaysia Sabah in the Sabah province. The disease severity was about 100% with 20% incidence. Red spots were primarily found on the leaf surfaces. Anthracnose developed as the disease progressed, and acervuli were observed in the spots. Small pieces of infected leaves (5 x 5 mm) were excised from spot margins, surface sterilized based on Khoo et al. (2022a), placed on potato dextrose agar (PDA) in Petri dishes, which were incubated for 5 days at 25°C in the dark. The colonies formed on the PDA plates were abundant with gray-white fluffy mycelia after 5 days, and the reverse view revealed brown. UMS01, a representative isolate, was used to morphologically and molecularly characterize the fungus. Conidia were one-celled, cylindrical, hyaline, smooth, and blunt at the ends, measuring 13.8 to 16.5 x 3.6 to 6.7 µm (n = 20). Appressoria ranged in size from 7.6 to 9.3 x 5.5 to 6.9 µm (n= 20) and were ovoid to clavate, spherical to irregular in shape and dark brown in color. Genomic DNA was extracted from fresh mycelia of isolate UMS01 based on Khoo et al. (2021) with the addition of mechanical disruption using a micro pestle before heating at 95°C. PCR amplification was performed based on Khoo et al. (2022a) using ITS1/ITS4, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and GDF1/GDR1 primer pairs to amplify the internal transcribed spacer (ITS) region, calmodulin (CAL), actin (ACT), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Weir et al. 2012). PCR products with positive amplicons were sent to Apical Scientific Sdn. Bhd. for sequencing. The sequences were deposited in GenBank under accession numbers OK458683 (ITS), OL953033 (CAL), OL953030 (ACT), OL953036 (CHS-1), and OL953039 (GAPDH). Before BLAST, the search set were adjusted to exclude model sequences (XM/XP) and the uncultured/environmental sample sequences, and limit to sequences from type material. They were 99-100% similar to the Colletotrichum siamense ITS (JX010171), CAL (JX009714), ACT (FJ907423) and CHS-1 (JX009865), and Colletotrichum changpingense GAPDH (MZ664048) type sequences. The GAPDH marker did not reliably resolve the relationships within the C. gloeosporioides complex (Vieira et al. 2020). Phylogenetic analysis using maximum likelihood based on the combined ITS, CAL, ACT, CHS-1 and GAPDH indicated that the isolate formed a supported clade (100% bootstrap value) to the most related C. siamense. Morphological and molecular characterization matched the description of C. siamense (Huang et al. 2021). Pathogenicity tests were performed to fulfil Koch's postulates by spraying a spore suspension (106 spores/ml) on the leaves of three healthy four-month-old 'Pokok Bakung' plants, while three additional plants were sprayed with water as a control. The inoculated plants were covered with plastics for 48 h at 25°C in the dark. Incubation was performed based on Iftikhar et al. (2022). Symptoms similar to those of the field collection occurred after 6 days post inoculation. No symptoms occurred on the control plants. The experiment was repeated two more times. The reisolated fungal isolates were identical to C. siamense morphologically and molecularly. Previously, C. siamense has been reported to cause anthracnose on Allamanda cathartica (Huang et al. 2021) and avocado (Li et al. 2022) in China, and 'Purple Dream' eggplant in Malaysia (Khoo et al. 2022b). Colletotrichum fructicola has been reported to cause anthracnose on C. asiaticum in China (Qing et al. 2020). To our knowledge, this is the first report of C. siamense causing anthracnose on C. asiaticum in Malaysia. Our findings expand the geographic range of C. siamense and indicate that it could be a potential threat limiting the growth and production of C. asiaticum in Malaysia.
'Purple Dream' eggplant (Solanum melongena) is widely grown for its edible fruits in Malaysia. In July 2021, anthracnose symptoms were observed on fruit with a disease severity of approximately 80% and an incidence of 10% in a field (14.6 m2) (5°56'50.9"N, 116°04'31.9"E) located in the Penampang district of Sabah province. The symptoms initially appeared as irregular light brown spots. As the disease progressed, the spots enlarged and merged into extensive lesion patches that appeared in concentric circles. The symptomatic fruit tissues (5 x 5 mm) were surface sterilized based on Khoo et al. (2022), and plated onto potato dextrose agar (PDA), and incubated at 25°C in the dark. Colonies with gray-white fluffy mycelia developed after 7 days, and the reverse of the colonies was dark brown. A representative isolate named Penampang was characterized morphologically and molecularly. The conidia were one-celled, cylindrical, blunt at the ends, hyaline, smooth, and measured 13.3 to 16.1 x 3.9 to 6.0 µm (n= 20). Appressoria ranged in size from 7.6 to 9.3 m x 5.5 to 6.6 µm (n= 20) and were spherical to irregular in shape and dark brown in colour. Genomic DNA was extracted from fresh mycelia of isolate Penampang based on Khoo et al. (2021) and Khoo et al. (2022). ITS1/ITS4, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and GDF1/GDR1 primer pairs were used to amplify the isolate's internal transcribed spacer region (ITS), and partial calmodulin (CAL), actin (ACT), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase genes (GAPDH) (Weir et al. 2012). PCR products were sequenced by Apical Scientific Sdn. Bhd. (Selangor, Malaysia). Sequences were deposited in GenBank under the accession numbers OL957466 (ITS), OL953035 (CAL), OL953032 (ACT), OL953038 (CHS-1), and OL953041 (GAPDH). They were 99% to 100% identical to the Colletotrichum ti ITS (NR_120143) (515 bp out of 519 bp), and C. siamense CAL (JX009714) (729 bp out of 731 bp), ACT (JX009518) (282 bp out of 282 bp), CHS-1 (JX009865) (299 bp out of 299 bp), and GAPDH (JX009924) (276 bp out of 277 bp) sequences. ITS sequences do not reliably resolve relationships within the C. gloeosporioides complex (Weir et al. 2012). The phylogenetic maximum likelihood analysis using the combined ITS, CAL, ACT, CHS-1, and GAPDH sequences indicated that the isolate was part of the C. siamense clade (100% bootstrap value) that also contained the type isolate ICMP 18578 of this species. Morphological and molecular characterization matched the description of C. siamense (Huang et al. 2021; Ismail et al. 2021). Koch's postulates were performed similarly as described by Chai et al. (2017) but using spray-inoculation (108 spores/ml) of three healthy 'Purple Dream' eggplant fruit with isolate Penampang. Water was sprayed on three additional fruits that served as controls. All the fruits were incubated at 25°C and less than 90% relative humidity. Symptoms similar to those observed in the field developed 5 days after inoculation. No symptoms occurred on controls. The experiment was repeated two more times. The reisolated fungi were identical to the pathogen morphologically and molecularly. To our knowledge, this is the first report of C. siamense causing anthracnose on fruit of 'Purple Dream' S. melongena in Malaysia as well as worldwide. Our findings expand the host range of C. siamense and indicate that the pathogen could potentially limit 'Purple Dream' eggplant production in Malaysia.
Production of watermelon (Citrullus lanatus) in Malaysia was 150,000 mt in 2020 (Malaysian Department of Agriculture, 2021). In November 2019, nine locally produced watermelon fruit (red flesh, seedless) from five local stores in the states of Kelantan, Terengganu, and Penang exhibited sunken, circular, brown lesions that enlarged to1.5 to 10 cm in diameter with scattered orange masses of conidia. Lesions coalesced to cover approximately 50% of the fruit surface. Lesions were surface sterilized by spraying 70% alcohol onto the fruit followed by drying with sterilized paper towels. A total of 153 tissue segments (1×1 cm) were excised from the rind, immersed in 1% sodium hypochlorite for 3 min, rinsed twice for 1 min in sterilized distilled water, air-dried, transferred to potato dextrose agar (PDA) plates, and incubated at 25±1°C for 7 days. Single-spore transfers produced pure cultures, resulting in 12 isolates. Colonies on PDA were initially white and turned pale gray with age. Conidia were hyaline, one end round and the other narrowly acute, aseptate, smooth-walled, straight, cylindrical to clavate, 10.5-16.5 µm × 3-4.5 μm (n = 30). Observed morphological characters matched published description of Colletotrichum spp. (Damm et al. 2012). Internal transcribed spacer (ITS) and glyceraldehyde-phosphate dehydrogenase (GAPDH) genes were amplified using primer sets ITS1/ITS4 and GDF1/GDF2, respectively. All sequences were deposited in GenBank (MW856808 for ITS; MZ219296 for GAPDH). A BLASTn search of both sequences on GenBank showed 99% identity with C. scovillei along with other closely related Colletotrichum species. Phylogenetic analysis of ITS and GAPDH alignments, using maximum likelihood along with reference strains of closely related species from Mycobank, confirmed species identity as C. scovillei. A pathogenicity test was conducted on two healthy watermelon fruit (red flesh, seedless). A 6-mm-diameter mycelial plug of a colony on PDA was positioned on a 0.5-cm-long wound on each fruit; a sterile PDA plug placed on a similar wound on the opposite side served as a control. Fruit were incubated at 25±1°C for 7 days in plastic-wrapped trays above distilled water to maintain high humidity. Small, sunken, circular brown lesions appeared and expanded at inoculation sites within 7 days. Symptoms were identical to those produced by natural infections, and the controls were asymptomatic. Isolates from the lesions at the inoculation sites were confirmed as C. scovillei based on morphological characteristics, fulfilling Koch's postulates. The pathogenicity test was conducted four times with a total of eight fruit. Many species in the C. orbiculare complex cause watermelon anthracnose (Keinath, 2018). To our knowledge, this is the first report of C. scovillei (C. acutatum species complex; Damm et al. 2012) causing anthracnose on watermelon in Malaysia. Anthracnose caused by C. scovillei has been confirmed on other crops such as pepper (Toporek and Keinath, 2021), banana (Zhou et al., 2017), and chili (Oo et al., 2017). This insight will inform efforts to improve management of watermelon anthracnose in Malaysia.
Ixora chinensis (family Rubiaceae), locally known as 'Bunga Jejarum', is widely grown as an ornamental shrub and as sources for phytochemicals with medicinal properties in Malaysia. In May 2021, irregular brown spots were found on the leaves of some 'Bunga Jejarum' in Universiti Malaysia Sabah (6°02'01.0"N 116°07'20.2"E) located in Sabah province. As the disease progressed, the spots enlarged and coalesced into large necrotic areas giving rise to drying of infected leaves. The disease severity was about 70% with 20% incidence. Five symptomatic leaves (5 x 5 mm) from five plants were excised and sterilized based on Khoo et al. (2022) before plated on five potato dextrose agar (PDA) and cultured at 25°C. After 5 days, white to pale honey and dense mycelia with lobate edge were observed on all PDA plates. Globose, black conidiomata semi-immersed on PDA were observed after a week. Two to four hyaline filamentous appendages 7.7 to 17.6 μm long attached to fusoid conidia (11.8 to 20.9 x 5.7 to 7.6 μm, n = 20), which consisted of a hyaline apical cell, basal cell, and three versicolored median cells. The upper two median cells were dark brown, while the lowest median cell was pale brown. The isolate of the causal pathogen was characterized molecularly. Genomic DNA of isolate UMS01 was extracted based on Khoo et al. (2021) and Khoo et al. (2022). Amplification of the internal transcribed spacer (ITS), tubulin (TUB) and translation elongation factor 1-α (TEF) region was performed based on Khoo et al. (2022) using primers ITS1/ITS4 (White et al. 1990), T1/Bt2b (Glass and Donaldson, 1995; O'Donnell and Cigelnik, 1997) and EF1-728/EF2 (O'Donnell et al. 1998; Carbone and Kohn, 1999), respectively. PCR products with positive amplicons were sent to Apical Scientific Sdn. Bhd. for sequencing. The isolate's sequences were deposited in GenBank as OM320626 (ITS), OM339539 (TUB) and OM339540 (TEF). They were 99% to 100% identical to ITS(KM199347) (545 out of 545 bp), TUB (KM199438) (768 out of 769 bp) and TEF (KM199521) (480 out of 481 bp) of the type sequences (CBS 600.96). Phylogenetic analysis using the maximum likelihood method based on the combined ITS, TEF and TUB sequences placed the isolate UMS01 in the same clade as the isolate CBS 600.96 of Neopestalotiopsis cubana. Thus, the pathogen was identified as N. cubanabased on the morphological description from Pornsuriya et al. (2020), molecular data in Genbank database and multigene sequence analysis. To further confirm its pathogenicity, the first and second leaves of three 'Bunga Jejarum' plants were inoculated by pipetting 1 ml aliquots of a 1 × 106 conidia/ml spore suspension. Three additional 'Bunga Jejarum' plants were mock inoculated by pipetting 1 ml of sterile distilled water on similar age leaves. The plants were covered with plastic bags after inoculation for 48 h before placing them in a glasshouse under room temperature. The leaves were sprayed with water to keep the leaf surfaces moist along the experiment. The incubation and disease observation were performed based on Chai et al. (2017) and Iftikhar et al. (2022). After 7 days post-inoculation, all infected leaves exhibited the symptoms observed in the field, whereas the controls showed no symptoms. The same fungus was isolated from the diseased leaves and, thus confirmed Koch's postulates. The experiment was repeated two more times. The reisolated fungi were visually and genetically identical to the original isolate obtained from the field samples. To our knowledge, this is the first report of N. cubana causing leaf blight on 'Bunga Jejarum' in Malaysia, as well as the world. Our finding has broadened the distribution and host range of N. cubana, indicating that it poses potential damage to the medicinal plant Bunga Jejarum in Malaysia.
Anthracnose fruit rot caused by various Colletotrichum spp. is a serious disease for pepper (Capsicum annuum) growers, resulting in extensive fruit loss (Harp et al. 2008). Samples of five pepper fruits were obtained from two commercial farms in Lexington and Pickens counties, South Carolina, in August and September 2019, respectively. All fruits had two or more soft, sunken lesions covered with salmon-colored spore masses. Pieces of diseased tissue cut from the margins of lesions were surface disinfested in 0.6% sodium hypochlorite, rinsed in sterile deionized water, blotted dry, and placed on one-quarter-strength potato dextrose agar (PDA/4) amended with 100 mg chloramphenicol, 100 mg streptomycin sulfate, and 60.5 mg mefenoxam (0.25 ml Ridomil Gold EC) per liter. Two isolates of Colletotrichum sp. per fruit were preserved on dried filter paper and stored at 10º C. One additional isolate of Colletotrichum sp. had been collected from a jalapeño pepper fruit on a farm in Charleston County, South Carolina, in 1997. Colony morphology of three isolates, one per county, on Spezieller Nährstoffarmer Agar (SNA) was pale grey with a faint orange tint. All isolates readily produced conidia on SNA with an average length of 16.4 μm (std. dev. = 1.8 μm) and a width of 2.2 μm (std. dev. = 0.2 μm). Conidia were hyaline, smooth, straight, aseptate, cylindrical to fusiform with one or both ends slightly acute or round, matching the description of C. scovillei (Damm et al. 2012). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-tubulin (TUB2) genes from three isolates were amplified and sequenced with the primer pairs GDF1/GDR1 and T1/Bt2b, respectively. Species within the C. acutatum clade can be readily distinguished with GAPDH or TUB2 (Cannon et al. 2012). The GAPDH and TUB2 sequences for all three isolates were 100% similar to each other and strain CBS 126529 (GAPDH accession number JQ948597; TUB2 accession number JQ949918) of C. scovillei (Damm et al. 2012). GAPDH and TUB2 sequences for each isolate were deposited in GenBank under the accessions MT826948-MT826950 and MT826951-MT826953, respectively. A pathogenicity test was conducted on jalapeño pepper fruits by placing a 10-ul droplet of a 5 x 105 conidial suspension of each isolate onto a wound made with a sterile toothpick. Control peppers were mock inoculated with 10 ul sterile distilled water. A humid chamber was prepared by placing moist paper towels on the bottom of a sealed crisper box. Inoculated peppers were placed on upside-down 60 ml plastic condiment cups. Three replicate boxes each containing all four treatments were prepared. The experiment was repeated once. After 7 days in the humid chamber at 26ºC, disease did not develop on control fruits, whereas soft, sunken lesions covered with salmon-colored spores developed on inoculated fruits. Lesions were measured and C. scovillei was re-isolated onto amended PDA/4 as previously described. Lesion length averaged 15.6 mm (std dev. = 4.1 mm) by 11.5 mm (std dev. = 2.0 mm). Colletotrichum sp. resembling the original isolate were recovered from all inoculated fruit, but not from non-inoculated fruit. C. scovillei has been reported in Brazil in South America and in China, Indonesia, Japan, Malaysia, South Korea, Taiwan, and Thailand in Asia (Farr and Rossman 2020). This is the first report of C. scovillei as the casual organism of anthracnose fruit rot on pepper in South Carolina and the United States.
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.
Rubber tree (Hevea brasiliensis (Willd. ex Adr. Juss) Müll. Arg.) is used for the extraction of natural rubber and is an economically and socially important estate crop commodity in many Asian countries such as Indonesia, Malaysia, Thailand, India, Sri Lanka, China and several countries in Africa (Pu et al, 2007). Xishuangbanna City and Wenshan City are the main rubber cultivation areas in Yunnan Province, China. In November 2012, rubber tree showing typical wilt symptoms (Fig. 1 A) and vascular stains (Fig. 1 B) were found in Mengla County, Xishuangbanna City. This disease was destructive in these trees and plant wilt death rate reached 5%. The diseased wood pieces (0.5cm long) from trunk of rubber was surface disinfected with 75% ethanol for 30s and 0.1% mercuric chloride (HgCl2) for 2min, rinsed three times with sterile distilled water, plated onto malt extract agar medium (MEA), and incubated at 28℃. After 7 days, fungal-like filaments were growing from the diseased trunk. Six cultures from 6 rubber trunk were obtained and incubated on MEA at 28℃, after 7 days to observe the cultural features. The mycelium of each culture was white initially on MEA, and then became dark green. Cylindrical endoconidia apices rounded, non-septate, smooth, single or borne in chains (8.9 to 23.6 × 3.81 to 6.3μm) (Fig. 1 C). Chlamydospores (Fig. 1 D) were abundant, thick walled, smooth, forming singly or in chains (11.1 to 19.2 × 9.4 to 12.0μm). The mould fungus was identifed as Chalaropsis based on morphology (Paulin-Mahady et al. 2002). PCR amplification was carried out for 3 isolates, using rDNA internal transcribed spacer (ITS) primer pairs ITS1F and ITS4 (Thorpe et al. 2005). The nucleotide sequences were deposited in the GenBank data base and used in a Blast search of GenBank. Blast analysis of sequenced isolates XJm8-2-6, XJm8-2 and XJm10-2-6 (accessions KJ511486, KJ511487, KJ511489 respectively) had 99% identity to Ch. thielavioides strains hy (KF356186) and C1630 (AF275491). Thus the pathogen was identified as Ch. thielavioides based on morphological characteristics and rDNA-ITS sequence analysis. Pathogenicity test of the isolate (XJm8-2) was conducted on five 1-year-old rubber seedlings. The soil of 5 rubber seedlings was inoculated by drenching with 40 ml spore suspension (106 spores / ml). Five control seedlings were inoculated with 40 ml of sterile distilled water. All the seedlings were maintained in a controlled greenhouse at 25°C and watered weekly. After inoculated 6 weeks, all the seedlings with spore suspension produced wilt symptoms, as disease progressed, inoculated leaves withered (Fig. 1 E) and vascular stains (Fig. 1 F) by 4 months. While control seedlings inoculated with sterile distilled water remained healthy. The pathogen re-isolated from all inoculated symptomatic trunk was identical to the isolates by morphology and ITS analysis. But no pathogen was isolated from the control seedlings. The pathogenicity assay showed that Ch. thielavioides was pathogenic to rubber trees. Blight caused on rubber tree by Ceratocystis fimbriata previously in Brazil (Valdetaro et al. 2015), and wilt by Ch. thielavioides was not reported. The asexual states of most species in Ceratocystis are "chalara" or "thielaviopsis" (de Beer et al. 2014). To our knowledge, this is the first report of this fungus causing wilt of rubber in China. The spread of this disease may pose a threat to rubber production in China.
Weeds may act as inoculum reservoirs for fungal pathogens that could affect other economically important crops (Karimi et al. 2019). In February 2019, leaves of the ubiquitous invasive weed, Parthenium hysterophorus L. (parthenium weed) exhibiting symptom of blight were observed at Ladang Infoternak Sg. Siput (U), a state-owned livestock center in Perak, Malaysia. Symptoms appeared as irregularly shaped, brown-to-black necrotic lesions across the entire leaf visible from both surfaces, and frequently on the older leaves. The disease incidence was approximately 30% of 1,000 plants. Twenty symptomatic parthenium weed leaves were collected from several infested livestock feeding plots for pathogen isolation. The infected tissues were sectioned and surface-sterilized with 70% ethyl alcohol for 1 min, rinsed three times with sterile distilled water, transferred onto potato dextrose agar, and incubated at 25°C under continuous dark for 7 days. Microscopic observation revealed fungal colonies with similar characteristics. Mycelium was initially white and gradually changed to pale orange on the back of the plate but later turned black as sporulation began. Conidia were spherical or sub-spherical, single-celled, smooth-walled, 12 to 21 μm diameter (mean = 15.56 ± 0.42 μm, n= 30) and were borne on a hyaline vesicle. Based on morphological features, the fungus was preliminarily identified as Nigrospora sphaerica (Sacc) E. W. Mason (Wang et al. 2017). To confirm identity, molecular identification was conducted using isolate 1SS which was selected as a representative isolate from the 20 isolates obtained. Genomic DNA was extracted from mycelia using a SDS-based extraction method (Xia et al. 2019). Amplification of the rDNA internal transcribed spacer (ITS) region was conducted with universal primer ITS1/ITS4 (White et al. 1990; Úrbez-Torres et al. 2008). The amplicon served as a template for Sanger sequencing conducted at a commercial service provider (Apical Scientific, Malaysia). The generated sequence trace data was analyzed with BioEdit v7.2. From BLASTn analysis, the ITS sequence (GenBank accession number. MN339998) had at least 99% nucleotide identity to that of N. sphaerica (GenBank accession number. MK108917). Pathogenicity was confirmed by spraying the leaf surfaces of 12 healthy parthenium weed plants (2-months-old) with a conidial suspension (106 conidia per ml) collected from a 7 day-old culture. Another 12 plants served as a control treatment and received only sterile distilled water. Inoculation was done 2 h before sunset and the inoculated plants were covered with plastic bags for 24 h to promote conidial germination. All plants were maintained in a glasshouse (24 to 35°C) for the development of the disease. After 7 days, typical leaf blight symptoms developed on the inoculated plants consistent with the symptoms observed in the field. The pathogen was re-isolated from the diseased leaves and morphological identification revealed the same characteristics as the original isolate with 100% re-isolation frequency, thus, fulfilling Koch's postulates. All leaves of the control plants remained symptomless and the experiment was repeated twice. In Malaysia, the incidence of N. sphaerica as a plant pathogen has been recorded on several important crops such as watermelon and dragon fruit (Kee et al. 2019; Ismail and Abd Razak 2021). To our knowledge, this is the first report of leaf blight on P. hysterophorus caused by N. sphaerica from this country. This report justifies the significant potential of P. hysterophorus as an alternative weed host for the distribution of N. sphaerica. Acknowledgement This research was funded by Universiti Putra Malaysia (UPM/GP-IPB/2017/9523402). References Ismail, S. I., and Abd Razak, N. F. 2021. Plant Dis. 105:488. Karimi, K., et al. 2019. Front Microbiol. 10:19. Kee, Y. J., et al. 2019. Crop Prot. 122:165. Úrbez-Torres, J. R., et al. 2008. Plant Dis. 92:519. Wang, M., et al. 2017. Persoonia 39:118. White, T. J. et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Xia, Y., et al. 2019. Biosci Rep. 39:BSR20182271.
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.
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.
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.
Fusarium wilt disease incited by Fusarium oxysporum f. sp. niveum (FON) is the utmost devastating soil-inhabiting fungal pathogen limiting watermelon (Citrullus lanatus) production in Malaysia and globally. The field disease survey of fusarium wilt was carried out during December 2019 and November 2020, in three major production areas (3 farmer fields per location) in Peninsular Malaysia namely, Mersing, Serdang and Kuantan and disease incidence of 30 and 45%, was recorded for each year, respectively. Infected watermelon plants showed symptoms such as vascular discoloration, brown necrotic lesions to the soil line or the crown, one-sided wilt of a plant, or a runner or the whole plant. Infected root and stem tissues, 1-2 cm pieces were surface sterilized with 0.6% NaOCl for 1 minute followed by double washing with sterile water. The disinfected tissues were air-dried and transferred onto semi-selective Komada's medium (Komada 1975) and incubated for 5 days. The fungal colonies produced were placed on potato dextrose agar (PDA) to attain a pure culture and incubated at 25±2℃ for 15 days. The pure fungal colony was flat, round and light purple in color. Macroconidia were straight to slightly curved, 18.56-42.22 µm in length, 2.69-4.08 µm width, predominantly 3 septate and formed in sporodochia. Microconidia measured 6.16-10.86 µm in length and 2.49-3.83 µm in width, kidney-shaped, aseptate and were formed on short monophialides in false-heads. Chlamydospores were single or in pairs with smooth or rough walls, found both terminally or intercalary. To confirm their pathogenicity, two-week-old watermelon seedlings (cv. NEW BEAUTY) were dipped into spore suspension (1 ˟ 106 spores/ml) of representative isolates of JO20 (Mersing), UPM4 (Serdang) and KU41 (Kuantan) for 30 second and then moved into 10 cm diameter plastic pots containing 300 g sterilized soil mix. Disease symptoms were assessed weekly for one month. Control seedlings were immersed in sterile distilled water before transplanting. The inoculated seedlings showed typical Fusarium wilt symptoms like yellowing, stunted growth, and wilting, which is similar to the farmer field infected plants. However, the seedlings inoculated by sterile distilled water remained asymptomatic. The pathogen was successfully re-isolated from the infected seedlings onto Komada's medium, fulfilling the Koch's postulate. For the PCR amplification, primers EF-1 and EF-2 were used to amplify the tef1-α region. A Blastn analysis of the tef1-α sequences of the isolates JO20 (accession nos. MW315902), UPM4 (MW839560) and KU41 (MW839562) showed 100% similarity; with e-value of zero, to the reference sequences of F. oxysporum isolate FJAT-31690 (MN507110) and F. oxysporum f. sp. niveum isolate FON2 790-2 (MN057702). In Fusarium MLST database, isolates JO20, UPM4 and KU41 revealed 100% identity with the reference isolate of NRRL 22518 (accession no. FJ985265). Though isolate FJ985265 belongs to the f. sp. melonis, earlier findings had revealed Fusarium oxysporum f. sp. are naturally polyphyletic and making clusters with diverse groups of the Fusarium oxysporum species complex (O'Donnell et al. 2015). The isolates JO20, UPM4 and KU41 were identified as F. oxysporum f. sp. niveum based on the aligned sequences of tef1-α and molecular phylogenetic exploration by the maximum likelihood method. To the best of our knowledge, this is the first report of F. oxysporum f. sp. niveum as a causative pathogen of Fusarium wilt disease of watermelon in Malaysia. Malaysia enables to export watermelon all-year-round in different countries like Singapore, Hong-Kong, The United Arab Emirates (UAE), and Netherlands. The outburst of this destructive soil-borne fungal pathogen could cause hindrance to watermelon cultivation in Malaysia. Thus, growers need to choice multiple management tactics such as resistant varieties, cultural practices (soil amendments and solarization), grafting, cover crops and fungicide application to control this new pathogen.
Repeated sampling conducted from December 2019 to March 2020, and fruit of pineapple (Ananas comosus) var MD2 showing early stem end rot symptoms including brown and rotten fruit skin near the stem end region (Fig.1Aa) or darker skin with black discoloration (Fig.1Ab) indicated a consistent fungal infection. The samples (30 fruits from each location) were collected from store houses in three farmer fields with 60% disease incidence in Serdang, (3.0220oN,101.7055oE), Selangor, West Malaysia. The pulp of infected fruits appeared watery with characteristic spoilage odour. Symptomatic necrotic tissues from stem end region and skin were cut in to pieces (1x1cm), surface sterilized and plated onto potato dextrose agar amended aseptically with 0.5 g L-1 streptomycin sulphate. The plates were incubated at room temperature (28±2oC) in natural light conditions. Five days old cultures were light grey in colour and gradually turned dark brown to black with dense deeply tufted, mycelium as the culture aged (Fig.1B, C). Conidial morphology was observed using compound microscope (Olympus model BX-50F4, Tokyo, Japan) equipped with Dino-Eye. Branched mycelia with 0-1 septate arthospores were evident in 14 days old cultures (Fig.1D). Measured arthroconidia (5 to10x3 to 4.5µm) were ellipsoid to ovoid or round shaped, hyaline with an acutely rounded apex, truncate base, initially aseptate (Fig.1E) and arranged as chain at maturity (Fig.1F). The pathogen was identified through PCR amplification of the internal transcribed spacer (ITS) region using ITS1 and ITS4 primers (White et al., 1990) and BLASTn homology search as Neoscytalidium dimidiatum based on 100% similarity to a reference sequence (accession number KJ648577) that was previously deposited (Mohd et al.,2013). The sequence was deposited in Gen Bank ( accession number MW082810). Pathogenicity test was performed using the mycelial plug inoculation method and repeated twice with five replicates. Healthy MD2 pineapple fruits were surface sterilized with 1% NaOCl solution for15 min. followed by washing with sterilized distilled water. One centimeter diameter PDA plug at the margin of actively growing seven days old cultures were inserted in each of two inoculation wounds made on the skin and stem end of each fruit then the wounds were wrapped with moist cotton wool. Non-colonized PDA plugs were used to inoculate the control fruits. Fruits were incubated under 85% RH at room temperature. Five days after inoculation, the fruits showed similar dark necrotic discoloration and confirmed as N.dimidiatum by PCR (Fig.1G). The Koch postulates were fulfilled by inoculation and re-isolation of the fungal pathogen. This pathogen has also been reported previously to cause economic losses on a number of other hosts, such as pitayah fruits in Israel and Malaysia (Erza et al., 2013; Mohd et al., 2013)) and almond in California (Mohomed et al., 2018). To our knowledge this is the first report of N. dimidiatum causing postharvest stem end rot on MD2 pineapple in Malaysia. It may have the possibility to develop postharvest economic losses to pineapple industry, if severely affected fruits with high population of the pathogen left unattended in store houses.
Snake gourd (Trichosanthes cucumerina L.), an annual climbing plant belonging to the family of Cucurbitaceae, is native to Southeast Asia countries, e.g., India, Pakistan, Malaysia, China, and Indonesia. It is commonly consumed as a vegetable and also used as a traditional herbal medicine due to the antidiabetic, anti-inflammatory, antibacterial, hepatoprotective, and cytotoxic activities (Devi 2017). In September 2020, phytoplasma-induced disease symptoms such as little leaf, yellowing, phyllody, virescence, and witches' broom were observed on snake gourd in Yunlin County, Taiwan. The cross-sectional examination of the symptomatic plant by transmission electron microscopy showed typical phytoplasma-like pleomorphic bodies with spherical, oval and tubular shapes in sieve elements. Further examination by nested PCR revealed that a 1.2 kb DNA fragment for 16S rRNA gene was only amplified from symptomatic leaf of snake gourd using the phytoplasma universal primer pairs P1/P7 followed by R16F2n/R16R2. BLAST and iPhyClassifier (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi) analyses on the amplified DNA fragment (accession no. MW309142) revealed that it shares 100% identity with that of GenBank accession NZ_AMWZ01000008 (complement [31109 to 32640]) of peanut witches' broom (PnWB) phytoplasma, a 'Candidatus phytoplasma aurantifolia'-related strain (Firrao et al. 2004), and could be classified into the 16SrII-V subgroup. Samples examined by nested PCR were further characterized by western blotting using the polyclonal antibody raised against the Imp of PnWB phytoplasma (Chien et al. 2020a, b). An expected signal of 19 kDa specific for Imp was only detected in the symptomatic snake gourd, but not in healthy snake gourd. Since the disease symptoms caused by phytoplasma infection are highly dependent on the secreted effectors (Namba 2019), phyllogen gene that is responsible for phyllody and virescence symptoms was amplified from symptomatic snake gourd by PCR. BLAST analysis revealed that phyllogen identified in snake gourd is identical with that of PnWB phytoplasma. In Taiwan, species of family Cucurbitaceae such as loofah, bitter gourd, and pumpkin are commonly infected by 16SrVIII phytoplasma (Davis 2017). In this study, we report for the first time that snake gourd, a species of family Cucurbitaceae, was infected by 16SrII-V PnWB phytoplasma in Taiwan.