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.
Dragon fruit or pitahaya (Hylocereus spp.) is a tropical fruit belonging to the Cactaceae. It is native to Central and South America and commercially grown in the United States in southern California, south Florida and Puerto Rico. During a disease survey from April to June 2020, stem canker was observed in greenhouses and commercial orchards located in Mayaguez and San Sebastian, Puerto Rico with an incidence of 80%. Diseased cladodes (stems) of 1 mm2 tissue sections of 23 pitahaya varieties (NOI-13, NOI-14, NOI-16, N97-15, N97-17, N97-18, N97-20, N97-22, American Beauty, Cosmic Charlie, Halley's comet, Purple Haze, Alice, Bloody Mary, Dark Star, David Bowie, Delight, Makisupa, Red Jaina, Soul Kitchen, Vietnamese Jaina, Neitzel and Lisa) were disinfested with 70% ethanol, rinsed with double distilled water and plated on potato dextrose agar (PDA) amended with 60 mg/L streptomycin. Three isolates (17B-173-T3, 12C-118-T1 and 13B-131-T2) of Neoscytalidium dimidiatum (syn. N. hyalinum) were identified using taxonomic keys (Crous et al., 2006) and sequencing of the internal transcribed spacer (ITS) with primers ITS5 and ITS4 (White et al. 1990) and translation elongation factor 1 alpha (TEF1-α) with primers EF1-728F and EF1-986R (Carbone and Kohn, 1999). Sequences were compared using the BLASTn tool with N. dimidiatum deposited in NCBI GenBank. In PDA, colonies of N. dimidiatum were initially powdery white and turned grayish-black with age. Arthroconidia (n=50) were dark brown, disarticulating, truncate or cylindrical at the base, thick-walled with 0 to 1 septum, averaging 9.1 X 5.5um in length. GenBank accession numbers of N. dimidiatum DNA sequences were MT921260, MT921261 and MT921262 for ITS and MT920898, MT920899 and MT920900 for TEF1-α. Sequences were 99-100% identical with Ex-isotype CBS145.78 accession numbers KF531816 for ITS and KF531795 for TEF1-α. Pathogenicity tests were conducted on 12 healthy dragon fruit plants of 1.5 years old using three non-detached cladodes per plant. Cladodes were inoculated with 5mm mycelial plugs from 8-day-old pure cultures grown on PDA. Three healthy dragon fruit plants were used as controls and were inoculated with PDA plugs only. The experiment was repeated once. Twenty days after inoculations (DAI), isolates of N. dimidiatum caused stem canker on dragon fruit plants. For all isolates, sunken orange spots averaged 3 X 2 mm in length at 8 DAI. Necrotic blotches with chlorotic halos averaged 10 X 15 mm at 14 DAI; stem cankers with water-soaked tissue were observed at 20 DAI, and arthroconidia and black pycnidia on dry stem cankers at 30 DAI. Untreated controls had no symptoms of stem canker, and no fungi were isolated from tissue. Neoscytalidium dimidiatum has been reported to cause stem canker on Hylocereus spp. in China, Florida, Israel, Malaysia and Taiwan (Chuang et al. 2012; Lan et al., 2012; Ezra et al., 2013; Sanahuja et al., 2016). To our knowledge, this is the first report of N. dimidiatum causing stem canker on dragon fruit in Puerto Rico. References: 1. Carbone, I., and Kohn, L. 1999. Mycologia, 91:553. doi:10.2307/3761358 2. Chuang, M. F. et al. 2012. Plant Disease 96: 906. https://doi.org/10.1094/PDIS-08-11-0689-PDN. 3. Crous, P. W., et al. 2006. Stud. Mycol. 55:235. https://doi.org/10.3114/sim.55.1.235 4. Ezra et al. 2013. Plant Disease 97: 1513. https://doi.org/10.1094/PDIS-05-13-0535-PDN 5. Lan, G.B. et al. 2012. Plant Disease 96: 1702. https://doi.org/10.1094/PDIS-07-12-0632-PDN 6. Sanahuja et al. 2016. Plant Disease 100: 1499. https://doi.org/10.1094/PDIS-11-15-1319-PDN 7. White, T., Bruns, T., Lee, S., and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.
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.
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.
Pears (Pyrus pylifolia L.) are cultivated nationwide as one of the most economically important fruit trees in Korea. At the end of October 2019, bleeding canker was observed in a pear orchard located in Naju, Jeonnam Province (34°53'50.54″ N, 126°39'00.32″ E). The canker was observed on trunks and branches of two 25-year-old trees, and the diseased trunks and branches displayed partial die-back or complete death. When the bark was peeled off from the diseased trunks or branches, brown spots or red streaks were found in the trees. Bacterial ooze showed a rusty color and the lesion was sap-filled with a yeasty smell. Trunks displaying bleeding symptoms were collected from two trees. Infected bark tissues (3 × 3 mm) from the samples were immersed in 70% ethanol for 1 minute, rinsed three times in sterilized water, ground to fine powder using a mortar and pestle, and suspended in sterilized water. After streaking each suspension on Luria-Bertani (LB) agar, the plates were incubated at 25°C without light for 2 days. Small yellow-white bacterial colonies with irregular margins were predominantly obtained from all the samples. Three representative isolates (ECM-1, ECM-2 and ECM-3) were subjected to further characterization. These isolates were cultivated at 39 C, and utilized (-)-D-arabinose, (+) melibiose, (+)raffinose, mannitol and myo-inositol but not 5-keto-D-gluconate, -gentiobiose, or casein. These isolates were identified as Dickeya sp. based on the sequence of 16S rRNA (MT820458-820460) gene amplified using primers 27f and 1492r (Heuer et al. 2000). The 16S rRNA sequences matched with D. fangzhongdai strain ND14b (99.93%; CP009460.1) and D. fangzhongdai strain PA1(99.86%; CP020872.1). The recA, fusA, gapA, purA, rplB, and dnaX genes and the intergenic spacer (IGS) regions were also sequenced as described in Van der wolf et al. (2014). The recA (MT820437-820439), fusA (MT820440-820442), gapA (MT820443-820445), purA (MT820446-820448), rplB (MT820449-820451), dnaX (MT820452-820454) and IGS (MT820455-820457) sequences matched with D. fangzhongdai strains JS5, LN1 and QZH3 (KT992693-992695, KT992697-992699, KT992701-992703, KT992705-992707, KT992709-992711, KT992713-992715, and KT992717-992719, respectively). A neighbor-joining phylogenetic analysis based on the concatenated recA, fusA, gapA, purA, rplB, dnaX and IGS sequences placed the representative isolates within a clade comprising D. fangzhongdai. ECM-1 to 3 were grouped into a clade with one strain isolated from waterfall, D. fangzhongdai ND14b from Malaysia. Pathogenicity test was performed using isolate ECM-1. Three two-year-old branches and flower buds on 10-year-old pear tree (cv. Nittaka), grown at the National Institute of Horticultural and Herbal Science Pear Research Institute (Naju, Jeonnam Province in Korea), were inoculated with 10 μl and 2 μl of a bacterial suspension (108 cfu/ml), respectively, after wounding inoculation site with a sterile scalpel (for branch) or injecting with syringe (for flower bud). Control plants were inoculated with water. Inoculated branches and buds in a plastic bag were placed in a 30℃ incubator without light for 2 days (Chen et al. 2020). Both colorless and transparent bacterial ooze and typical bleeding canker were observed on both branches and buds at 3 and 2 weeks post inoculation, respectively. No symptoms were observed on control branches and buds. This pathogenicity assay was conducted three times. We reisolated three colonies from samples displaying the typical symptoms and checked the identity of one by sequencing the dnaX locus. Dickeya fangzhongdai has been reported to cause bleeding canker on pears in China (Tian et al. 2016; Chen et al. 2020). This study will contribute to facilitate identification and control strategies of this disease in Korea. This is the first report of D. fangzhongdai causing bleeding canker on pears in Korea.
Natural rubber is an important industrial raw material and an economically important perennial in China. In recent years, A new leaf fall disease, caused by Neopestalotiopsis aotearoa Maharachch., K.D. Hyde & Crous, has occurred in Indonesia, Malaysia, Thailand, Sri Lanka, and other major rubber planting countries. In May and July of 2020, this disease was first found on 2-year-old rubber seedlings in two plantations located in Ledong and Baisha counties in Hainan Province, China. In the two plantations of approximately 32 ha, 15% of the rubber seedlings had the disease and the defoliation was more than 20%. The infected leaves turned yellow and watery, and dark brown and nearly round lesions of 1-2 mm in diameter were formed on the leaves. When the humidity was high, the center of the lesion was grey-white, and the lesions had many small black dots, black margins and surrounded by yellow halos. When the disease was severe, leaves fell off. To identify the pathogen, leaf tissues were collected from lesion margins after leaf samples were surface-sterilized in 75% ethanol, rinsed with sterile water for three times, and air dried. The leaf tissues were plated on potato dextrose agar (PDA) and incubated at 28°C for seven days. Fungal cultures with similar morphology were isolated from 90% of tested samples and two isolates (HNPeHNLD2001 and HNPeHNLD2002) were used in pathogenicity and molecular tests. Rubber leaves (clone PR107) were inoculated with conidial suspension (106 conidia/ml), and inoculated with PDA were used as the control, Each treatment had 3 leaves, and each leaf was inoculated with 3 spots and incubated at 28oC under high moisture conditions. Five days later, leaves inoculated with conidial suspension showed black leaf spots resembling the disease in the field, whereas the control leaves remained symptomless. The fungal cultures isolated from the inoculated tissues, had identical morphology compared with the initial isolates. Colonies on PDA were 55-60 mm in diameter after seven days at 28°C, with undulate edges, pale brown, thick mycelia on the surface with black, gregarious conidiomata; and the reverse side was similar in color. Black conidia were produced after eight days of culture on PDA. Conidia were fusoid, ellipsoid, straight to slightly curved, 4-septate, ranged from 18.35 to 27.12 μm (mean 22.34 μm) × 4.11 to 7.03 μm (mean 5.41 μm). The basal cells were conic with a truncate base, hyaline, rugose and thin-walled, 4.35 to 6.33 μm long (mean 4.72 μm). Three median cells were doliform, 12.53 to 18.97 μm long (mean 15.26 μm), hyaline, cylindrical to subcylindrical, thin- and smooth-walled, with 2-3 tubular apical appendages, arising from the apical crest, unbranched, filiform, 14.7 to 25.3 μm long (mean 19.94 μm). The basal appendages were singlar, tubular, unbranched, centric, 3.13 to 7.13 μm long (mean 5.48 μm). Morphological characteristics of the isolates were similar to the descriptions of N. aotearoa (Maharachchikumbura et al. 2014). The rDNA internal transcribed spacer (ITS) region, translation elongation factor 1-αgenes (TEF), and beta-tubulin (TUB2) gene were amplified using the primer pairs ITS1/ITS4, EF1-728F/EF1-986R and T1/Bt-2b (Pornsuriya et al. 2020), respectively. The sequences of these genes were deposited in GenBank (ITS Accession Nos.: MT764947 and MT764948; TUB2: MT796262 and MT796263; TEF: MT800516 and MT800517). According to the latest classification of Neoprostalotiopsis spp. (Maharachchikumbura et al. 2014) and multilocus phylogeny, isolates HNPeHNLD2001 and HNPeHNLD2002 were clustered in the same branch with N. aotearoa. Thus, the pathogen was identified as N. aotearoa, which is different from N. cubana and N. formicarum reported in Thailand (Pornsuriya et al. 2020; Thaochan et al. 2020). The Neopestalotiopsis leaf spotdisease of rubber tree (H. brasiliensis) was one of the most serious and destructive leaf diseases in major rubber planting countries in Asia. ( Tajuddin et al. 2020) The present study of leaf fall disease on rubber tree caused byN. aotearoa is the first report in China. The finding provides the basic pathogen information for further monitoring the disease and its control.
In February 2022, leaf zonate spot disease afflicted Aloe vera L. in Yunnan, China, endangering the $39 billion industry with 0.33ha under cultivation (Wan 2015). The disease manifested with watery spots progressing into oval or circular necrosis lesions, characterized by a dark center surrounded by a gray-brown zone. In the late stage of the disease, lesions regress in size and several small dark picnidia dots appeared on the gray-brown zone. The disease incidence ranged from 10% to 15% in three commercial plantations. If left uncontrolled, the disease could diminish the commercial value of Aloe vera plants. Eighteen symptomatic leaf samples underwent morphological and genetic identification. The samples were carefully washed with distilled water and 1×1 cm2 sections of tissue were excised using a sterile scalpel. The sections underwent surface-disinfection with 3% NaOCl for 3 min and 75% ethanol for 30 s. After three sterile water rinses the sections were air-dried. Subsequently, they were transferred to potato dextrose agar (PDA) before being incubated at 25 ℃ in the dark. Of the 18 samples, eight produced the colonies with similar morphological characteristics, named LH7. Isolate LH7 had downy to woolly aerial mycelia, initially pinkish white on the surface, and gradually turned greenish-olivaceous from the middle, and eventually turned dark brown to black after seven days. The fungus formed arthric chains in the aerial mycelium on PDA but did not produce conidiomata. The conidia, which occurred in arthric chains were 5.50-9.9 × 4.08-7.51 μm (mean 7.09× 5.26 μm, n=50) in size, cylindrical, brown, and 0-1 septate. To ascertain LH7's pathogenicity, three healthy one-year old aloe plants were surface-sanitized with a 1% aqueous chlorine solution, rinsed with sterile water, and dried. Three leaves from each plant were punctuated and inoculated using conidial suspension (100 μl of 1x 106 conidial mL-1), while three control plants were inoculated with sterile distilled water. The pathogenicity tests were repeated twice. The inoculated plants were kept at 25 ℃ with a 12-hour light/12-hour dark cycle. After seven days, symptoms observed in the field appeared in the plants, while no disease occurred in the control plants. After 21 days, conidiomata formed on the inoculated leaves, averaging 116.92 μm (n=20) in diameter. These conidiomata were globose to subglobose, and brown to sub-brown. The fungus was successfully re-isolated from symptomatic tissue and the resulting colonies were morphologically consistent with isolate LH7. Based on the characteristics, the fungus was identified as Neoscytalidium dimidiatum (Philips et al. 2013). The specimen was deposited in China Center for Type Culture Collection ( CCTCC AF 2024001). This identification was confirmed through sequencing of ITS gene region of rDNA using ITS1/ITS4 (Imran et al. 2022). The sequence was submitted into GenBank database (ON878059). BLAST analysis of the LH7's ITS amplicon showed 100% similarity with that of JN093303.1. A phylogenetic tree constructed using the maximum likelihood method revealed that ON878059 was clustered with JN093303.1. Previous studies have documented that pathogens such as Colletotrichum gloeosporioides (Penz.), Fusarium spp. and Rhizopus oryzae can also cause diseases in A. vera in China (Zhou et al. 2008; Ding et al. 2015). Additinonally, Cladosporium sphaerospermum, Pseudopestalotiopsis theae, and Lasiodiplodia theobromae have been identified as causal agents of aloe leaf spot diseases in India, Bangladesh and Malaysia (Avasthi et al. 2016; Ahmmed et al. 2022; Khoo et al. 2022). To our knowledge, this is the first report of N. dimidiatum causing leaf necrosis of aloe in China. Vigilant surveillance and disease control measures are imperative to mitigate potential losses in this region.
Aloe vera L. var. chinensis (Haw.) Berg. (family Asphodelaceae), locally known as 'Lidah Buaya', is an economically important plant as the gel from the leaves possesses anti-inflammatory, anti-arthritic, antibacterial, and hypoglycemic properties and is used for cosmetic, pharmaceutical and healing purpose in Malaysia. In July 2021, irregular black sunken spots (3- to 10-mm in diameter) were observed on the leaves of 'Lidah Buaya' plants under leaf development stage in the field located in the district Penampang of Sabah province (N5°56'37.1" E116°04'21.5"). The disease severity was about 30% with 10% incidence. The tissues surrounding the black spots became brown and dry when the plants grew older. No gel contained in the sunken zones. Symptomatic leaf tissues (5 x 5 mm) were cut from the infected margin, surface sterilised with 75% ethanol for 1 minute, washed with 2% sodium hypochlorite solution for 1 minute, rinsed, and air dried before plating on five potato dextrose agar (PDA) plates (pH 7). Plates were incubated at 25°C for 3 days in the dark. Greyish-white fluffy mycelia were observed, and then became dark grey with age. Dark pigmentation in each plate was produced after a week of incubation at 25°C. A representative isolate Penampang was further characterized morphologically and molecularly. Immature conidia were single-celled, aseptate, ellipsoid and hyaline, measuring 19.4 × 24.5 µm (n = 30). Mature conidia were brown, thick-walled and one-septate with longitudinal striations, 22.5 × 28.3 µm (n = 30). Genomic DNA was extracted from fresh mycelia of isolate Penampang based on the extraction method described by Khoo et al. (2021) with additional of mechanical disruption using micro pestle before heating. KOD One PCR master mix (Toyobo, Japan) containing hot-start modified KOD DNA polymerase was used for PCR amplification. The PCR condition were 94°C for 10 s, 55°C for 5 s and 72°C for 2 s, for 30 cycles, and initial denaturation of 94°C for 3 min and a final extension step of 72°C for 5 min. The internal transcribed spacer (ITS) region of rDNA and tubulin (TUB) genes were amplified using ITS1/ITS4 and T10/Bt2b primer sets, respectively (O'Donnell et al. 1997; White et al. 1990). The products were then sent to Apical Scientific Sdn. Bhd. for sequencing. The generated ITS (OK209451) and TUB (OL660667) were 100% identical to L. theobromae isolate MRR-161 and CPC:27690 (GenBank MW282884 and MT592639, respectively) in BLASTn analysis. Phylogenetic analysis using maximum likelihood based on the combined ITS and TUB sequences indicated that the isolates formed a supported clade (91% bootstrap value) to the related L. theobromae. The morphological and molecular characterization of the fungus matched L. theobromae described by Pečenka et al. (2021). Mycelial agar plugs (5-mm-diameter) from 7-day-old PDA culture of Penampang isolate were placed onto pinpricked leaves of three 2-month-old 'Lidah Buaya' plants. Pinpricked leaves of three 2-month-old 'Lidah Buaya' plants received sterile 5-mm-diameter PDA agar plugs to serve as controls. The inoculated 'Lidah Buaya' plants were covered with plastics for 48 h, and were incubated at 25°C. All inoculated leaves developed symptoms as described above 6 to 7 days post-inoculation, whereas no symptoms occurred on controls, thus fulfilling Koch's postulates. The experiments were repeated twice. The reisolated fungus was identical to representative isolate Penampang morphologically and molecularly. L. theobromae was reported previously on A. vera in Cuba (Urtiaga 1986) and India (Mathur 1979). To our knowledge, this is the first report of L. theobromae causing leaf spot on A. vera in Malaysia. The occurrence of this disease emphasizes the importance of disease surveillance in the region. Plant disease management strategies need to be established to reduce the losses.
Multiple diseases, including brown spot (Cochliobolus miyabeanus), leaf spot (Epicoccum sorghimum), and blast (Magnaporthe oryzae), can cause spot-like symptoms on the leaves of rice. In July 2021, a disease showing symptoms like brown spot was observed in an 8-hectare field of rice, with disease incidence of >30%, in Beaumont, Texas. Lesions started as small pinhead-size blackish spots on leaf tips or from the edges of leaf blades. The spots enlarged to become irregular (most) or oval brown spots with a slight chlorotic halo. Diseased leaves were collected, washed in running tap water and cut into small pieces. Pieces of the tissue were surface sterilized with 1%NaOCl for 2 min followed by 75% ethanol for 30 s and then washed in sterile distilled water three times with each time lasting for 1 min. The disinfected tissue pieces were air dried, placed on potato dextrose agar (PDA) medium and incubated at 25℃. Initially fungal colonies were hairy in texture with light dark brown center and whitish edge and dark brown pigmentation at the reverse side. Mature colonies turned to black in the center and dark brown toward the edge, with black at the reverse side after 2 or more weeks of incubation. Conidia were oval to narrowly oblong, rounded at the ends, with 2 to 6 distoseptate, and 15 to 35 × 6 to 10 µm in size. These morphological characteristics were similar to those described for Curvularia hawaiiensis (Aslam et al. 2019; Ellis 1971; Kusai et al. 2015). For molecular identification, DNA was extracted and the two different rRNA regions internal transcribed spacer (ITS) and large subunit (LSU), and the two genes RNA Polymerase II (RPB1) and translation elongation factor 1 alpha (EF1) of the fungus were amplified using the primers of ITS1/ITS4 (Wang et al. 2014), D1/D2 domain region of LSU (Fell et al. 200), and RPB1 and EF1 (Wang et al. 2014), respectively, and sequenced. The ITS sequence (OK397200) was 98.27% identical to C. hawaiiensis (KP131943); the EF1 sequence (OK492159) was 99.78% identical to C. hawaiiensis (KC503942); the LSU sequence (OK397295) was 98.96% identical to multiple C. hawaiiensis (MN055715, MH160813, MH875853, etc.); the RPB1 sequence (OK492160) was 97.41% identical to C. hawaiiensis (JN992363). To evaluate pathogenicity, three rice plants (cv. Presidio) at the 3-leaf stage were spray inoculated with a conidial suspension of 1 x 106 conidia/ml. Another set of three plants that were sprayed with sterilized distilled water served as the controls. Treated plants were maintained in a greenhouse with temperature ranging from 25 to 30℃. After 2 weeks, typical symptoms, like those observed in the field, developed on the inoculated plants while no symptoms developed on the control plants. The same fungus was consistently re-isolated from the diseased plants. The pathogenicity test was conducted three times with similar results. To our knowledge, this is the first report of brown leaf spot caused by C. hawaiiensis in rice in the United States. Curvularia species are frequently associated with rice grain and cause blackish discoloration symptoms on grain kernels. Recently, however, C. hawaiiensis has also been reported to cause brown leaf spot in Malaysia (Kusai et al. 2015) and Pakistan (Aslam et al. 2019). This research will help identify this disease from other leaf spot-like diseases and develop effective management strategies.
Watermelon (Citrullus lanatus) accounts for almost 13% of all tropical fresh fruit production in Malaysia. They are grown, mostly in Johor, Kedah, Kelantan, Pahang, and Terengganu areas of Malaysia on 10,406 ha and yielding 172,722 Mt. In 2019, a new fruit rot disease was observed in two major production areas in Peninsular Malaysia. Disease symptoms included water-soaked brown lesions on the fruit surface in contact with the soil. The lesions enlarged gradually and ultimately covered the whole fruit with white mycelium leading to internal fruit decay. Disease surveys were conducted in December 2019 and November 2020 in fields at Kuantan, Pahang and Serdang, Selangor. Disease incidence was 10% in 2019 and 15% in 2020. Infected fruits were collected and washed under running tap water to wash off adhering soil and debris. Fruit tissue sections 1 to 2 cm in length were surface sanitized with 0.6% sodium hypochlorite (NaOCl) for 3 min. and washed twice with sterile distilled water. The disinfected air-dried tissues were then transferred onto potato dextrose agar (PDA) media and incubated at 25±2℃ for 3 days. Fungal colonies with whitish mycelium and pink pigment isolated using single spore culture. The pure cultures were placed onto carnation leaf agar (CLA), and the culture plates were incubated at 25±2℃ for 15 days for morphological characterization. On CLA, macroconidia were produced from monophialides on branched conidiophores in orange sporodochia. Macroconindia were thick-walled, strong dorsiventral curvature, 5 to 7 septate with a tapered whip-liked pointed apical cell and characteristic foot-shaped basal cell, 21.9 to 50.98 μm long and 2.3 to 3.60 μm wide. Typical verrucose thick chlamydospores with rough walls were profuse in chains or clumps, sub-globose or ellipsoidal. Based on morphological characteristics they were identified as Fusarium equiseti (Leslie and Summerell 2006). Molecular identification of both U4-1 and N9-1 pure culture isolates were carried out using two primer pair sets; internal transcribed spacer (ITS) ITS-1/ ITS-4 and translation elongation factor 1 alpha (TEF1-α) (EF-1/EF-2). A Blastn analysis of the ITS gene sequence of U4-1(MW362286) and N9-1 (MW362287) showed >99% similarity index to the reference gene sequence of F. equiseti isolate 19MSr-B3-4 (LC514690). The TEF1-α sequences of U4-1 (accession no. MW839563) and N9-1 (accession no. MW839564) showed 100% identity; with an e-value of zero, to the reference gene sequence of F. equiseti isolate URM: 7561 (accession no. LS398490). Each isolate also had a >99% identity with isolate NRRL 34070 (accession no. GQ505642) in Fusarium MLST database that belongs to the F. incarnatum-equiseti species complex (O'Donnell et al. 2015). Based on phylogenetic analysis of the aligned sequences (TEF1-α) by the maximum likelihood method, the U4-1 and N9-1 isolates were confirmed to be F. equiseti as was reported in Georgia, USA (Li and Ji 2015) and in Harbin, Heilongjiang Province, China (Li et al. 2018). Finally, the two pure culture isolates of U4-1 and N9-1 were used to fulfill Koch's postulates. Stab inoculations of five healthy watermelon fruits (cv. 345-F1 hybrid seedless round watermelon) were performed with a microconidial suspension of individual isolates (4x106 spores/mL). Five control fruits were stabbed with double distilled water. The inoculated fruits were incubated under 95% relative humidity at a temperature of 25±2℃ for 48 h followed by additional incubation inside an incubator at 25±2℃ for 8 days. Ten days post-inoculation, the control fruits showed no disease symptoms. However, inoculated fruits exhibited typical symptoms of fruit rot disease like water-soaked brown lesions, white mycelium on the fruit surface and internal fruit decay, which is similar to the farmer's field infected fruits. The suspected pathogen was successfully re-isolated from the symptomatic portion of inoculated fruit and morphologically identified for verification. To our knowledge, this is the first report of F. equiseti causing fruit rot of watermelon in Malaysia. Malaysia exports watermelon year-round to many countries around the world. The outbreak of this new fruit rot disease could potentially pose a concern to watermelon cultivation in Malaysia.
Rice (Oryza sativa L.) has been farmed in Malaysia since ancient times and is one of the most important commercial crops (Ma'arup et al. 2020). Throughout January to August 2022, chlorotic spots with brown halos ranging 2 to 10 mm wide were found on upper leaves of rice variety Mahsuri in the vegetative stage with a severity and incidence of approximately 60% and 100%, respectively in Kampung Tagas, Sabah, Malaysian Borneo (06°09'41.8"N, 116°13'45.1"E). As the disease developed, the spots coalesced into larger chlorotic spots. Three leaf pieces (5 x 5 mm) were excised from lesion margins, surface sterilized based on Khoo et al. (2022a), before plating on water agar (WA) at 25°C. Purification of fungi was conducted on WA using hyphal tip isolation. When three pure cultures were obtained, the fungi were cultured on potato dextrose agar (PDA) and WA for 7 days in 12 h light and 12 h dark at 25°C for the macro- and micro-morphological characterization, respectively. The colonies of the three isolates on PDA were initially gray, later becoming dark. Conidia (n=30) were fusiform, smooth-walled, dark-brown, and melanized with three transverse septa, measuring 7.3 to 11.4 × 16.2 to 27.2 µm. The isolates were named Tagas01, Tagas02, Tagas03. Genomic DNA was extracted from fresh mycelia of the pathogens based on the extraction method described by Khoo et al. (2022b). The primers ITS1/ITS4 (White et al. 1990), GPD1/GPD2 (Berbee et al. 1991), and EF1-983F/EF1-2218R (Schochet al. 2009) were used to amplify the internal transcribed spacer (ITS) region of rDNA, partial fragments of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor (EF-1α) region, respectively based on PCR conditions as described previously (Khoo et al. 2022a). The sequences were deposited in GenBank under accession numbers OP268402, OP271304, OP271305 (677/677 bp) (ITS), OP270699, OP270703, OP270704 (609/613 bp) (GAPDH), OP270700-OP270702 (928/930 bp) (EF-1α). They were 99.35-100% similar to the Curvularia lunata ITS (HF934911), GAPDH (LT715821), and Curvularia dactyloctenicola EF-1α (MF490858) type sequences. Although C. dactyloctenicolais related to C. lunata, the conidia of the former are much wider making them easier to differentiate (Marin-Felix et al. 2017). Phylogenetic analysis using maximum likelihood based on the combined ITS, GAPDH and EF-1α sequences indicated that the isolate formed a supported clade to C. lunata. The pathogens were identified as C. lunata based on morphological and molecular characterization. Koch's postulates were performed. Three replicate healthy rice at the vegetative stage were sprayed with a spore suspension of 1 × 106 spore/ml in distilled sterilized water, prepared from 1-week-old fungal culture, grown in the dark on WA. Three replicate rice plants were sprayed with distilled sterilized water as control. Plants were covered with transparent polyethylene bags to keep moisture, and kept in a greenhouse at ~27°C. Bags were removed after 4 days of incubation. Monitoring and incubation were performed in greenhouse based on Iftikhar et al. (2022). The pathogenicity test was also performed using isolate Tagas02 and Tagas03. All inoculated leaves developed symptoms as described after 6 days post-inoculation, whereas no symptoms occurred on controls. The experiments were repeated twice. The reisolated fungi were identical to the pathogen morphologically and molecularly, thus fulfilling Koch's postulates. C. lunata has been reported in Peninsular Malaysia (Lee et al. 2012). This is the first report of C. lunata causing leaf spot on Oryza sativa in Sabah, Malaysian Borneo. This illness not only reduces yields and lowers milling quality, but it may also be mistaken for rice blast, necessitating needless fungicide spraying.