'Thai Gold' yellow pitahaya (family Cactaceae, Selenicereus megalanthus) is a new crop being planted commercially in Malaysia. In May 2021, reddish-brown necrotic lesions were observed on the stems of approximately 60% of 'yellow pitahaya' plants in the field (~8 ha) located in the district Keningau of Sabah, Malaysia (5°20'53.1"N 116°06'23.0"E). As the disease progressed, the smaller lesions merged into larger irregularly shaped areas that formed dark brown in color. Stems with reddish-brown spot symptoms from ten plants were collected from the field and brought to the laboratory in sterilized paper bags. The symptom margin was excised into small blocks (5 x 5 x 5 mm). The blocks were surface sterilized based on Khoo et al. (2022), and placed on potato dextrose agar (PDA). The pathogens were isolated (three isolates were obtained) and cultured on potato dextrose agar (PDA) at 25°C for 5 days in the dark. The isolates developed floccose, white colony that darkened with age in PDA. Conidia (n = 30) were single celled, black, smooth, globose to subglobose, 13.9 to 18.7 μm in diameter, and borne singly on a hyaline vesicle at the tip of each conidiophore. Genomic DNA was extracted from fresh mycelia based on Khoo et al. (2021) and Khoo et al. (2022). Amplification of the internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-α (tef1-a) region and β-tubulin (tub2) genes were performed using ITS1/ITS4 (White et al. 1990), EF1-728F/EF2 (O'Donnell et al. 1998; Carbone and Kohn, 1999) and T10/Bt2b (Glass and Donaldson, 1995; O'Donnell and Cigelnik, 1997) primer sets, respectively. The products were sent to Apical Scientific Sdn. Bhd. for purification and sequencing. BLASTn analysis of the newly generated ITS (OK448496, OM832586, OM832589) were 100% identical to Nigrospora sphaerica isolate 1SS (MN339998) (507/507 bp), tef1-a (OM223859, OM826971, OM826972) were 100% identical to Nigrospora sphaerica isolate F (MT708197) (497/497 bp) and tub2 (OL697400, OM826973, OM826974) were 100% identical to Nigrospora sphaerica isolate SN180517 (MN719407) (434/434 bp). The isolates established a supported clade to the related N. sphaerica type sequences, according to phylogenetic analysis using maximum likelihood based on the concatenated ITS, tef1-a and tub2 sequences. Morphological and molecular characterization matched the description of N. sphaerica (Kee et al. 2019). Koch's postulates were performed by spray inoculation (106 spores/ml) of isolate Keningau on the stem of three 'Thai Gold' yellow pitahaya plants in growth stage 4 (BBCH code: 419) (Kishore, 2016), while water was sprayed on three mock controls. The experiment was repeated using isolate Keningau02 and Keningau03 as inoculants. The inoculated stems on yellow pitahaya plants were covered with plastics for 48 h, and the plants were maintained in a greenhouse at room temperature 25 to 28°C with a relative humidity of 80 to 90%. All the inoculated stems developed symptoms 5 days post-inoculation, whereas no symptoms occurred on mock controls, thus fulfilling the Koch's postulates. No pathogen was isolated from the mock controls. The experiments were repeated two more times for each isolate. The reisolated fungi were identical to N. sphaerica morphologically and molecularly. Previously, N. sphaerica has been reported to cause stem brown spot disease on S. megalanthus in the Philippines (Taguiam et al. 2020). To our knowledge, this is the first report of N. sphaerica causing stem brown spot on 'Thai Gold' S. megalanthus in Malaysia. Our findings serve as a warning for the authorities and farmers that the disease threat has appeared for the Malaysian yellow pitahaya production.
Peru is the ninth exporter of coffee (Coffea arabica) in the world, and Amazonas is among its most important producing departments (INIA 2019). In July 2021, in the nursery of the "Instituto de Investigación para el Desarrollo Sustentable de Ceja de Selva", in Huambo district (6° 26' 11.19'' S; 77° 31' 24.18'' W), four-month-old coffee seedlings cv. Catimor with 0.5-2.0 cm brown concentric leaf spots and rotten stems, bearing white mycelial tufts and black sporodochia, were observed at 30% incidence. Infected seedlings were collected. Foliar sections of 2-3 mm with infected tissue were surfaced disinfected in 2% NaClO and transferred onto Petri plates containing potato dextrose agar medium (PDA). The plates were incubated at 25° C for 7 days. We obtained three isolates (INDES-AFHP61, INDES-AFHP62 and INDES-AFHP66) with similar morphology from different seedlings. Colonies (16-17 mm diam.) formed concentric rings with white aerial mycelium, giving rise to viscous and olivaceous dark green sporodochial conidiomata. Conidia (4.82-5.77 × 1.34-1.65 µm; n = 30) were cylindric, hyaline, smooth, and aseptate. These morphological features correspond to Paramyrothecium spp. (Lombard et al. 2016). The DNA of isolates was extracted using the Wizard® Purification Kit (Promega Corp., Madison, Wisconsin), and the internal transcribed spacer 1 and 2 intervening the 5.8S subunit rDNA region (Accession numbers: OM892830 to OM892832), and part of the second-largest subunit of the RNA polymerase II, the calmodulin and the β-tubulin genes (OM919453 to OM919461) were sequenced following Lombard et al. (2016). All sequences had a percent identity greater than or equal to 99% to corresponding sequences of the P. roridum type specimen (CBS 357.89). Additionally, a multilocus Maximum Likelihood phylogenetic analysis incorporating sequence data from previous relevant studies (Lombard et al. 2016; Pinruan et al. 2022) grouped our three isolates together with the type and other specimens of P. roridum in a strongly supported clade, confirming the species identification. To evaluate pathogenicity, four-month-old coffee seedlings cv. Catimor were sprayed with 10 mL of conidial suspensions at 1 x 106 /mL. A set of control seedlings were inoculated with sterile water. Seedlings were maintained in a humidity chamber at 25 °C. After 15 days brown concentric foliar spots, stem rotting, mycelial tufts and sporodochia (same symptoms and signs observed originally at the nursery) arose in the non-control seedlings. The pathogen was re-isolated on PDA, confirming P. roridum was the causal agent of leaf spot and stem rot diseases of coffee. Paramyrothecium roridum has wide geographic distribution and host range (Lombard et al. 2016). This pathogen was reported to infect C. arabica in Mexico and Coffea sp. in Colombia (Pelayo-Sánchez et al. 2017; Lombard et al. 2016; Huaman et al. 2021). It was also reported in Africa infecting soybeans (Haudenshield et al. 2018), in Brazil infecting Tectona grandis (Borges et al. 2018), in Egypt infecting strawberries (Soliman 2020), and in Malaysia infecting Eichhornia crassipes (Hassan et al. 2021). To the best of our knowledge, this is the first time P. roridum is reported on coffee in Peru.
Anisomeles indica (L.) Kuntze is a perennial erect herb that belongs to the genus Epimeredi, family Labiatae (Hsieh et al., 2008). This herb is distributed in several southern provinces such as Yunnan, Sichuan and Guizhou in China, and it is also exported to Southeast Asian countries such as Singapore and Malaysia (Li., 2010; Yao et al., 2019). Due to its market potential and broad development prospects, the herb has been cultivated in Yunnan. In August 2021, virus-like symptoms on leaves, including shrinking, mosaic, and yellow mottling(Fig S1. A) appeared on approximately 80% of A. indica in the experimental fields of the Kunming Institute of Botany, Chinese Academy of Science, in Kunming, Yunnan. To unveil the possible viral agents associated with the disease symptoms, leaf samples were collected from 5 plants for transmission electron microscopy (TEM) analysis using negative staining (Zhang et al., 2016). Rhabditiform-shaped particles around 300 × 18 nm (Fig S1. C) were observed, which resemble those of tobamoviruses. To identify the exact virus, total RNA was extracted from the 20 leaf samples using the RNA-easy Isolation Reagent (Vazyme, Nanjing, China), followed by reverse transcription (RT)-PCR with a degenerate tobamovirus primer pair (Li et al., 2014). A 480-bp amplicon was obtained from each sample and cloned into the pMD18-T vector for Sanger sequencing (Takara, Dalian, China). BLASTn-analysis revealed that the 20 amplicons were identical and shared 100% nucleotide sequence identity with tobacco mosaic virus (TMV) isolate Bei Cang Zhu from Atractylodes lancea (acc. no. KU198186) One sequence was deposited in the GenBank under the accession number OK489807. ELISA testing with TMV-specific antibody (Agdia, USA) produced positive results for all of the 20 leaf samples. In order to understand the difference between TMV isolates from A. indica and those form other host plants, the sequences of movement protein (MP, 807 bp) and RNA-dependent RNA polymerase (RdRp, 3351 bp) of TMV were also obtained from one of the TMV infected samples using the target gene special primers (Tab. S1), and submitted to GenBank under the accession number OM3662406 (MP) and OM366242 (RdRp). BLASTn-analysis revealed that the amplicon of MP shared 97.75% nucleotide sequence identity with TMV isolate Henan 9-2-2017 from sweet potato (MN186255.1) and RdRp shared 97.43% nucleotide sequence identity with TMV isolate SXFQ from Solanum lycopersicum (JX993906.1). Phylogenetic analysis indicated that the isolate of A. indica grouped with several TMV isolates (e.g., tomato, AF103779.1 and tobacco, HE818449.1) from Northern China. The virus was successfully transmitted onto healthy A.indica plants (n = 5) upon mechanical inoculation as the plants not only developed foliar distortion symptoms but also tested positive for TMV by RT-PCR with the CP-specific primers (Tab. S1). Taken together, our results demonstrated that the diseased A. indica plants were infected with TMV. To our knowledge, this is the first report of TMV infected A. indica (L.) Kuntze in China. Symptomatic phenotype-based field surveys on some plantations in Yunnan Province indicated that the disease incidence ranged from 70% to 90%, resulting in significant loss of production of A. indica. It is necessary to monitor the viruses in the fields and find effective methods to protect TMV in the A. indica (L.) Kuntze industry.
Tomato leaf curl New Delhi virus (ToLCNDV), a member of the genus Begomovirus in the family Geminiviridae is naturally transmitted by the whitefly Bemisia tabaci (order Hemiptera, family Aleyrodidae) in a circulative and persistent manner (Moriones et al. 2017). ToLCNDV has occurred in Bangladesh, India, Indonesia, Iran, Italy, Malaysia, Pakistan, Sri Lanka, Spain, Thailand and Tunisia (Moriones et al. 2017). To date, The primary cultivated host of ToLCNDV has been identified as tomato (Lycopersicon esculentum), but the virus is also known to infect 43 other plant species from a range of families including Cucurbitaceae, Euphorbiaceae, Solanaceae, Malvaceae and Fabaceae (Zaidi et al. 2017). In August 2021, virus-like symptoms including leaf deformation and curing were observed on tomato (Lycopersicon esculentum) in a greenhouse of about 0.5 hectares in Zhejiang Province, China. To identify viral agents potentially associated with this disease, an Oxford Nanopore cDNA library from a symptomatic sample was generated and sequenced. Total RNA was extracted using RNAiso Plus (TaKaRa, Tokyo, Japan). Libraries were constructed using Oxford Nanopore PCR-cDNA Sequencing Kit (SQK-PCS109; Oxford Nanopore Technologies, Oxford, UK), as recommended. Approximately 8.7 million reads were obtained from the Oxford MinION platform. After removing the adapters and low-quality reads, the clean reads were subjected to BLASTn analysis against the nt database. Approximately 797 and 168 reads produced high nt identities to the genome of ToLCNDV DNA-A (GeneBank Accession No. U15015.2) and ToLCNDV DNA-B (GeneBank Accession No. U15017.2) respectively. We designed 6 primer pairs (Table S1) to obtain the sequence of ToLCNDV Zhejiang (ToLCNDV-ZJ) isolate DNA-A and DNA-B. Briefly, total DNA from ToLCNDV-infected tomato was extracted using standard cetyl trimethylammonium bromide method. Segments of ToLCNDV DNA-A and DNA-B were amplified using high-fidelity DNA polymerase KOD-Plus-Neo (Toyobo, Osaka, Japan). PCR products were cloned into the pLB vector (Tiangen, Beijing, China) and Sanger sequenced. The obtained sequences were assembled into complete sequences of ToLCNDV-ZJ DNA-A (2,739 nt, GeneBank Accession No. OP356207) and DNA-B (2,693 nt, GeneBank Accession No. OP356208). Pairwise sequence comparison revealed that the ToLCNDV -ZJ shared the highest nt sequence identities of 98.7% and 98.4% with the genome segments of New Delhi isolate (genome A: HM159454) and India:Delhi:Cucumis:2012 isolate (genome B: KC545813) respectively. Furthermore, we performed PCR detection on 10 collected samples using the primer pair P1F and P1R. All eight symptomatic plants showing upward leaf curling and leaf distortion tested positive for ToLCNDV infection, whereas two asymptomatic plants were ToLCNDV free. To our knowledge, this is the first report of ToLCNDV infecting tomato in China, and with the widespread presence of B. tabaci in green houses, ToLCNDV may be a potential threat to the cultivation of tomato in China. In addition, ToLCNDV is an exceptional Old World bipartite begomovirus. In China, monopartite DNA satellite-associated begomoviruses with mostly narrow geographical ranges predominate, and are widespread (Li et al., 2022). The occurrence of ToLCNDV in China, which indicates that the success of this virus would become an emerging threat to vegetable and fiber crops.
Platostoma palustre (family Lamiaceae), locally known as 'Black Cincau', is an herb processed as herbal drinks in Malaysia. In November 2021, brown lesions were observed on leaf samples of P. palustre with an incidence of approximately 10% in a nursery in Penampang, Sabah province (5°55'30.4"N 116°04'35.7"E). The lesions developed into larger chlorotic spots with aging of leaves. Five samples of infected leaves were collected, excised (5 × 5 mm), and then surface sterilized with 75% ethanol for 1 minute, washed with 2% sodium hypochlorite solution for 1 minute, rinsed, and air dried before inoculated onto potato dextrose agar (PDA). Inoculated plates were incubated at 25°C. Three isolates were isolated from the samples, which showed cottony aerial mycelia with light purple concentric rings appeared on the reverse side of the colony after 3 days. Pycnidia which were spheroid and measured 64.0 to 114.1 × 41.2 to 88.0 μm (n= 30). Conidia, unicellular, hyaline, oval and measured 3.8 to 4.9 × 2.0 to 2.7 μm (n= 30). Chlamydospores were observed, either unicellular or multicellular. NaOH test on oatmeal agar positive, brownish red. Further, the genomic DNA of pathogens (UMS, UMS02 and UMS03) was extracted from fresh mycelia (7-day-old) using lysis buffer. Large Sub Unit (LSU), β-tubulin (tub) and RNA polymerase II (RPB2) gene were amplified using LR0R/LR7, T10/Bt2b and RPB2-5F2/RPB2-7cR primers (Rehner and Samuel, 1994; O'Donnell and Cigelnik, 1997; Liu et al. 1999) respectively. The sequences of isolate UMS, UMS02 and UMS03 which deposited in Genbank were OM238129, ON386254, ON386255 (LSU), OM048108, ON366806, ON366807 (tub), and ON003417, ON366804, ON366805 (RPB2). They had 99-100% homology to the LSU (1328/1328 bp) of Epicoccum sorghinum isolate Lido01 (OM501128), tub (422/425 bp) of isolate BJ-F1 (MF987525), and RPB2 (596/596 bp) of isolate HYCX2 (MK836295). Phylogenetic analysis by maximum likelihood method generated from the combined tub, LSU and RPB2 sequences indicated that the isolates formed a supported clade to the related Epicoccum sorghinum type sequences. Morphological, NaOH test and molecular characterization matched the description of E. sorghinum (Boerema et al. 2004; Li et al. 2020). Koch's postulates were performed by spray inoculation (106 conidia/mL) on the leaves of three healthy P. palustre seedlings with isolate UMS, while water was sprayed on three additional P. palustre seedlings served as controls. The plants were maintained in a greenhouse at room temperature 25 to 28°C with a relative humidity of 80 to 90%. All inoculated plants exhibited the symptoms similar to those of the nursery collection occurred after 8 days post inoculation. No symptoms occurred on controls. The experiment was repeated twice. The reisolated pathogen was morphologically identical to E. sorghinum. E. sorghinum was reported previously on Myrica rubra in China (Li et al. 2020). To our knowledge, this is the first report of E. sorghinum causing leaf spot on P. palustre in Malaysia. Our findings expand the host range of E. sorghinum in Malaysia.
Mango originated in the Indo-Burmese region (Alphonse de Candolle, 1885). In the Caribbean, Puerto Rico currently produces and exports mangoes to the United States and Europe. Globally, an important disease affecting mango production is dieback, caused by fungi belonging to Botryosphaeriaceae family. During a one-year survey from 2019 to 2020, conducted at the mango germplasm collection of the Agricultural Experiment Station of the University of Puerto Rico, located at Juana Díaz, PR, symptoms of dieback were observed in shoots, descending towards the woody part, and vascular necrosis. We sampled bimonthly, 35 Keitt trees for one year. At the end of the evaluation, we detected that a 74% disease incidence was caused by Botryosphaeriaceae. Lasiodiplodia mahajangana (syn. L. caatinguensis) was associated with 4% disease incidence. In addition, we identified other Botryosphaeriaceae species causing 70% of disease incidence. To identify the causal agent, sections of symptomatic tissue (4mm2) were surface disinfected by immersion in 70% ethanol, 10% sodium hypochlorite and rinsed with sterile-distilled water for 1 minute at each solution. Sections were transferred to petri dishes containing potato dextrose agar acidified with 85% lactic acid (aPDA). Ten fungal isolates were obtained with similar morphological characteristics such as colony color and texture, after 12 days. Of these, one representative (isolate 17) was selected and identified as L. mahajangana (Lm) using morphological parameters and sequences of four nuclear genes (Zhang, W. et al., 2021). In aPDA, Lm colonies showed sparse and slow-growing aerial mycelium with dark gray-greenish color at the center and light gray edges. Black pycnidia were observed after 15 days of incubation at 28°C and dark conditions. Hyaline, ovoid to ellipsoid immature conidia (n=40) with average size of 22 µm long and 12 µm wide were observed. Mature bicellular pigmented conidia (n=40) had longitudinal striate and its average size was 23 µm long and 12 µm wide. Internal transcribed spacer (ITS), β-tubulin (βtub), elongation factor 1-alpha (EF1-α) and large ribosomal subunit (LSU) genetic regions were amplified by PCR from the original and pathogenicity test recovered isolates. Sequences of PCR products were compared with NCBI database BLAST tool with other Lm sequences. Sequence accession numbers of the four genetic regions of Lm are as follows: OL375401 and OL375402 for the ITS region; OL405579 and OL405580 for β-tubulin; OL455922 and OL455923 for EF1-α; and OL375648 and OL375649 for LSU. All the sequences were grouped with the ex-type CMM1325 of Lm (BS=84). Pathogenicity tests were performed on 6-month-old mango trees of cv. Keitt. Three healthy trees were inoculated with 5 mm mycelial disks of Lm, on stems, with and without wounds. Controls were inoculated with aPDA disks only. Inoculated trees were covered for 3 days with plastic bags, keeping them in conditions of high relative humidity with constant irrigation, temperature of 28°C, and 12 hours of light and 12 hours of darkness for 12 days. Twelve days after inoculation, Lm isolates caused stem necrosis and canker, with differences in lesion severity from 2 to 17 mm2 with wound, and 0 to 6 mm2 without wound. Untreated controls showed no symptoms of canker. Lasiodiplodia mahajangana was re-isolated from diseased stems fulfilling Koch's postulates, and a sequence of the recovered isolate from the pathogenicity test was compared and included in the phylogenetic analysis. Lasiodiplodia mahajangana has been reported to cause stem-end rot of mango in Malaysia (Li, L. et. al., 2021). To our knowledge, this is the first report of Lm causing canker of mango in Puerto Rico. Knowing L. mahajangana as a new pathogen that causes canker of mango is important to establish an adequate and effective control management of this disease in mango producing countries worldwide.
Rice (Oryza sativa) is a staple food for most of the world's populations, particularly in Asia (Gumma et al. 2011). The rice sector provides Malaysians with a food supply, food sufficiency, and income for growers (Man et al. 2009). From January to February 2022, panicle samples showing symptoms of bacterial panicle blight (BPB) disease, including reddish-brown, linear lesions with indistinct margins on flag-leaf sheaths and blighted, upright, grayish straw-colored florets with sterile and aborted grains on panicles were collected in granary areas in Sekinchan, Selangor, Malaysia with 90% disease incidence in fields. Surface-sterilization of infected leaf tissue was performed using 75% ethanol and 1% sodium hypochlorite, followed by rinsing three times in sterilized water. Leaf tissue was macerated in sterilized water and aliquots were spread on King's B agar medium, then cultured for 24 h to 48 h at 35 °C. All isolated bacteria were Gram-negative rods, positive for catalase and gelatinase but negative for indole, oxidase and hydrogen sulfide (H2S), and utilized sucrose, inositol, mannitol, glucose, and citrate. Colonies were circular and smooth-margined, producing a diffusible yellowish-green pigment on King's B agar medium, which are characteristics of Burkholderia species (Keith et al. 2005). Five representative isolates (UPMBG7, UPMBG8, UPMBG9, UPMBG15, UPMBG17) were selected for molecular and pathogenicity tests. PCR using specific primers targeting the gyrB gene for molecular characterization was performed, and the ∼470 bp amplicons were sequenced (Maeda et al. 2006) and deposited in GenBank (OM824438 to OM824442). A BLASTn analysis revealed that the five isolates were 99% identical to the B. gladioli reference strains MAFF 302533, GRBB15041, and LMG19584 in GenBank (AB190628, KX638432, and AB220898). A phylogenetic tree using Maximum-likelihood analysis of the gyrB gene sequences showed that the five isolates were 99% identical to B. gladioli reference strains (AB190628, KX638432, and AB220898). To verify the identification of these isolates, the 16S rDNA gene was amplified using 16SF/16SR primers (Ramachandran et al. 2021), producing ~1,400 bp amplicons. The resulting sequences of the five isolates (OM869953 to OM869957) were 98% identical to the reference strains of B. gladioli (NR113629 and NR117553). To confirm pathogenicity, 10 ml suspensions of the five isolates at of 108 CFU/ml were inoculated into the panicles and crowns of 75-day-old rice seedlings of local rice varieties MR269 and MR219 grown in a glasshouse with temperatures ranging from 37 °C to 41 °C (Nandakumar et al. 2009). Control rice seedlings were inoculated with sterilized water. All isolates produced BPB disease symptoms like those originally found in the rice fields at four weeks after inoculation. Control seedlings remained asymptomatic. To fulfill Koch's postulates, the bacteria were reisolated from symptomatic panicles and were confirmed as B. gladioli by sequence analysis of the gyrB and 16S rDNA genes. To our knowledge, this is the first report of B. gladioli causing BPB disease of rice in Malaysia. Since BPB disease causes a significant threat to the rice industry, it is crucial to investigate the diversity of this destructive pathogen for effective disease management strategies in Malaysia.
Basella rubra (family Basellaceae), locally known as 'Remayong Merah', is an edible perennial vine served as leafy greens in Malaysia. In May 2021, leaves with circular brown spots ranging from 3 to 10 mm wide with purple borders were found on B. rubra growing in Penampang (5°56'55.6"N 116°04'33.5"E), Sabah province. The disease severity was 80% with 10% disease incidence on 50 plants. As the disease developed, the lesions grew larger and they developed necrotic centers. Leaves with brown spot symptoms from five plants were collected from the field. Five leaf pieces (5 x 5 mm) were excised from lesion margins, surface sterilized based on Khoo et al. (2022b), before incubation on water agar at 25°C. When five pure cultures were obtained, the fungi were cultured on potato dextrose agar (PDA) at 25°C. After 5 days, fluffy white mycelia tinged with pink pigmentation showing on the underside of the colony were observed on PDA. Mycelia became violet in color as the culture aged. The isolates were incubated on carnation leaf agar at 25°C with a 12-hour light/dark photoperiod for 10 days. Sickle-shaped, thin-walled and delicate macroconidia (n= 30), predominantly 3 septate, ranging from 21.6 to 38.3 μm long by 2.7 to 4.2 μm wide in size were observed. Kidney-shaped, aseptate microconidia (n= 30) ranged from 6.2 to 11 μm long by 2.6 to 3.9 μm wide in size, and were formed on monophialides in false heads. Chlamydospores were detected both terminally and intercalarily, singly or in pairs, with smooth or rough walls. Genomic DNA was extracted from fresh mycelia of a representative isolate from Penampang based on Khoo et al. (2022a). The primers ITS1/ITS4 (White et al. 1990) and EF1/EF2 (O'Donnell et al. 1998) were used to amplify the internal transcribed spacer (ITS) rDNA and translation elongation factor 1-α (TEF1α) region, respectively based on PCR conditions as described previously (Khoo et al. 2022b). The products were sent to Apical Scientific Sdn. Bhd. for sequencing. In BLASTn analysis, ITS sequence (OK469301) was 99% (506/507 bp) identical to isolate TSE07 (MT481761) of Fusarium oxysporum, and the TEF1α sequence (OM743433) was 100% (705/705 bp) identical to isolate BLBL5 of Fusarium oxysporum. The TEF1α sequence of Penampang was analyzed at the Fusarium MLST site (https://fusarium.mycobank.org/), and had 98% similarity to TEF1α of F. oxysporum (NRRL 22551). The pathogen was identified as F. oxysporum based on morphological (Leslie and Summerell 2006) and molecular data. A volume of 0.16 ml of spore suspensions (1 × 106 conidia/ml) were inoculated on a spot on each leaf of every three healthy B. rubra seedlings at the two-leaf stage. An additional three B. rubra seedlings were mock inoculated by pipetting sterile distilled water on similar aged leaf. The seedlings were maintained in a greenhouse at 25°C with a relative humidity of 80 to 90%. Six days after inoculation, all inoculated leaves exhibited the same symptoms as observed in the field, while the controls showed no symptoms. The experiment was repeated two more times. The reisolated fungi had the same morphology and DNA sequences as the original isolate obtained from the field samples, completing Koch's postulates. F. oxysporum has been reported previously in Bangladesh and India causing leaf spot disease on B. rubra (Dhar et al. 2015; Shova et al. 2020). To our knowledge, this is the first report of F. oxysporum causing leaf spot on B. rubra in Malaysia. The identification of leaf spot caused by F. oxysporum will enable plant health authorities and farmers to identify practices to minimize disease on this important crop.
Basella alba (family Basellaceae) is a perennial vine that serves as an edible leaf vegetable in Malaysia. In May 2021, red spots were observed on leaf samples of B. alba in Lido, Sabah Province (5°56'39.1"N, 116°04'47.6"E). The disease severity was about 20% with 10% incidence. The spots enlarged and coalesced into larger necrotic spots. Small pieces (5 x 5 mm) of infected leaves were excised from three plants, and then surface disinfected based on Khoo et al. (2022). One fungal isolate (Lido01) was isolated and cultured on potato dextrose agar (PDA) at 25°C. A single isolate with cottony aerial mycelia and pink concentric rings was observed on the upper surface of the culture. Unicellular and multicellular chlamydospores were observed, and measured 7.1 to 14.3. × 17.8 to 74.5 μm. Conidia were unicellular, hyaline, oval, and measured 3.8 to 5.2 x 1.7 to 2.7 μm (n= 20). Pycnidia were spheroid, and measured 66.2 to 114.3 x 44.1 to 86.1 μm (n= 20). Genomic DNA was extracted from fresh mycelia according to the extraction method of Khoo et al. (2022a and 2022b). ITS1/ITS4, LR0R/LR7, ACT512F/ACT783R, and T10/Bt2b primers were used to amplify the internal transcribed spacer (ITS), large subunit (LSU), actin (ACT), and tubulin (TUB) genes, respectively (O'Donnell and Cigelnik, 1997; Chen et al. 2021). PCR products were Sanger sequenced by Apical Scientific Sdn. Bhd. (Serdang, Malaysia). Sequences of isolate Lido01 were deposited in GenBank as OM501130 (ITS), OM501128 (LSU), OM513916 (ACT) and OM513917 (TUB). Respective gene sequences of this isolate showed 100% homology to ITS sequence of isolate BPL01 (OM453926) (507/507 bp), LSU sequence of isolate BPL01 (OM453925) (1328/1328 bp), ACT sequence of isolate CZ01 (MN956831) (275/275 bp) and TUB sequence of isolate BJ-F1 (MF987525) (556/556 bp). The sequences of Lido01 established a supported clade (99% bootstrap value) to the related Epicoccum sorghinum type sequences, according to phylogenetic analysis using maximum likelihood based on the concatenated ITS, ACT, and TUB sequences. Morphological characters also matched the description of E. sorghinum (Li et al. 2020). Koch's postulates were tested as described by Chai et al. (2017) with modification by spray inoculation (106 spores/ml) on the leaves of three healthy one-month-old B. alba, while sterilized distilled water served as the control treatment. Monitoring and incubation were performed in a greenhouse based on Iftikhar et al. (2022). All inoculated leaves developed symptoms as described above by 8 days post-inoculation, whereas no symptoms occurred on controls, thus fulfilling Koch's postulates. The experiment was repeated twice. The reisolated pathogen was morphologically and genetically identical to E. sorghinum. E. sorghinum was reported causing leaf spot on Brassica parachinensis (Yu et al. 2019), Camellia sinensis (Bao et al. 2019), Myrica rubra (Li et al. 2020), Oryza sativa (Liu et al. 2020) and Zea mays (Chen et al. 2021). To our knowledge, this is the first report of E. sorghinum causing leaf spot on B. alba in Malaysia. Our findings have expanded the geographic range and host range of E. sorghinum in Malaysia, though the host range of this isolate is not known.
Bothriochloa ischaemum (family Poaceae) is a perennial weed that can be found in borders of agricultural fields, pastures and roadsides in Malaysia. B. ischaemum is an important phytoremediation species in copper tailings dams (Jia et al. 2020). In December 2021, chlorotic spots with brown halos were observed on leaf samples of B. ischaemum with an incidence of approximately 80% in Penampang, Sabah province (5°56'50.4"N, 116°04'32.8"E). On older leaves, the spots coalesced into larger chlorotic spots. Small pieces (5 x 5 mm) of infected leaves collected from three plants were excised, and then surface sterilized according to Khoo et al. (2022). The fungus was isolated (one isolate was obtained) and cultured on potato dextrose agar (PDA) at 25°C. After 3 days, the colony had cottony aerial mycelia with light purple concentric rings appearing on the underside of the colony. Chlamydospores were produced, either unicellular or multicellular. Conidia were unicellular, hyaline, oval, and were 3.7 to 5.1 x 1.8 to 2.6 μm (n=20). Pycnidia were spheroid, and were 66.4 to 115.3 x 43.1 to 87.4 μm (n=20). Genomic DNA was extracted from fresh mycelia of the fungus based on the extraction method described by Khoo et al. (2022). Amplification of the internal transcribed spacer (ITS) region and large subunit (LSU) of rDNA, and actin (ACT), tubulin (TUB) and RNA polymerase II second largest subunit (RPB2) genes was performed using ITS1/ITS4, LR0R/LR7, ACT512F/ACT783R, T10/Bt2b and RPB2-5F2/RPB2-7cR primers, respectively (O'Donnell and Cigelnik, 1997; Liu et al. 1999; Sung et al. 2007; Chen et al. 2021). The PCR products were sequenced at Apical Scientific Sdn. Bhd.. Sequences were deposited in GenBank as OM453926 (ITS), OM453925 (LSU), OM451236 (ACT), OM451237 (TUB) and OM863567 (RPB2). Sequences of our isolate had 100% homology to ITS of isolate UMS (OK626271) (507/507 bp), LSU of isolate UMS (OM238129) (1328/1328 bp), ACT of isolate CZ01 (MN956831) (275/275 bp), TUB of isolate BJ-F1 (MF987525) (556/556 bp) and RPB2 of isolate HYCX2 (MK836295) (596/596 bp) sequences. Phylogenetic analysis was performed using the maximum likelihood method based on the general time reversible model with a gamma distribution and invariant sites (GTR + G + I) generated from the combined ITS, TUB, LSU and RPB2 sequences, indicating that the isolates formed a supported clade to the related Epicoccum sorghinum type sequences. Morphological and molecular characterization matched the description of E. sorghinum (Li et al. 2020). Koch's postulates were performed by spray inoculation (106 spores/ml) on the leaves of three healthy B. ischaemum plants, using isolate BPL01, while sterilized water was sprayed on three additional B. ischaemum which served as the control. Symptoms similar to those occurred after 6 days post inoculation. No symptoms occurred on controls. The experiment was repeated two more times. The reisolated pathogen was morphologically and genetically identical to E. sorghinum. E. sorghinum was reported previously on Brassica parachinensis (Yu et al. 2019), Camellia sinensis (Bao et al. 2019), Myrica rubra (Li et al. 2020), Oryza sativa (Liu et al. 2020) and Zea mays (Chen et al. 2021) in China. To our knowledge, this is the first report of E. sorghinum causing leaf spot on B. ischaemum in Malaysia. Our findings expand the geographic range and host range of E. sorghinum in Malaysia. B. ischaemum which is a weed in agricultural fields is a host of the pathogen and therefore could be a potential threat to Brassica parachinensis, Camellia sinensis, Oryza sativa and Zea mays in Malaysia. Weed management could be an effective way to eliminate inoculum sources of E. sorghinum.
Basella rubra (family Basellaceae), locally known as 'Remayong Merah', is the edible perennial vine served as leafy vegetable in Malaysia. In May 2021, B. rubra's leaves with circular to subcircular purple spots (ranging from 1-10 mm wide) were collected in Lido (5°56'44.6"N 116°04'46.5"E), Sabah province. The disease severity was about 60% with 20% disease incidence on fifty plants. As disease developed, the spots grew larger and necrosis were formed within the purple spots. Small pieces (5 x 5 mm) of five diseased spots were excised, and then surface sterilized based on Khoo et al. (2022b) before plating on water agar at 25°C. Once obtained the pure cultures from all diseased spots, they were incubated on potato dextrose agar at 25°C. After 7 days, white aerial mycelium with light violet pigmentation on lower side were observed on PDA. Then, the fungi were cultured on Carnation leaf agar (CLA) at 25°C and 12-h light/dark photoperiod for 10 days. Thin-walled slender and slightly curved macroconidia (n= 20) with 3 to 5 septa were ranged from 2.3 to 2.6 µm wide by 26.8 to 44.5 µm long in size. Oval microconidia (n= 20) with no septa were 2 to 2.2 µm wide by 9.5 to 15 µm long in size. Chlamydospores were absent. Monophialids with false head were observed. Isolate Lido and Lido02 were kept in the Laboratory of Genetics, Faculty of Science and Natural Resources, Universiti Malaysia Sabah for public request. Their genomic DNA were extracted from fresh mycelia of isolates based on Khoo et al. (2022a). EF1/EF2, RPB1-Fa/RPB1-G2R and RPB2-5f2/RPB2-7cr (Jiang et al. 2021) were used to amplify the translation elongation factor 1-α (TEF1) region, RNA polymerase largest subunit gene (RPB1) and RNA polymerase second largest subunit gene (RPB2) based on PCR condition in Khoo et al. (2022b). The isolate's sequences were deposited in GenBank as OM048109, OM634654 (TEF1), OM634655, OM634657 (RPB1) and OM634656, OM634658 (RPB2). They were 99 to 100% homology to TEF1 of isolate DPCT0102-2 (LC581453) (657/657 bp), RPB1 of strain ZJ05 (MT560605) (1558/1558 bp) and RPB2 of isolate GR_FP248 (MT305154) (1867/1869 bp) sequences. These sequences were polyphasic identified at the Fusarium MLST (https://fusarium.mycobank.org/), and were more than 99% similarity to Gibberella fujikuroi species complex (NRRL 25200). Gibberella fujikuroi and Fusarium fujikuroi are synonymous with Fusarium proliferatum (Leslie and Summerell 2006). The pathogen was identified as F. proliferatum based on morphological characterization, molecular data and phylogenetic analysis. Two non-wounded leaves of three one-month-old B. rubra seedlings were inoculated with mycelium plug (10 x 10 mm). Additional three B. rubra seedlings received sterile PDA agar plug (10 x 10 mm) to serve as controls. They were incubated in a glasshouse at room temperature 25°C with a relative humidity of 80 to 90%. After 8 days of inoculation, all inoculated leaves exhibited the symptoms as observed in the field, while the controls showed no symptoms, thus confirming the Koch's postulates. The experiment was repeated two more times. The reisolated pathogens were identified as F. proliferatum via PDA macroscopically, CLA microscopically and PCR amplification. F. proliferatum was reported previously causing leaf spot disease on Cymbidium orchids (Wang et al. 2018), tobacco (Li et al. 2017) and tomato (Gao et al. 2017). To our knowledge, this is the first report of F. proliferatum causing leaf spot on B. rubra in Malaysia. Infections of leaves reduce plant vigor and marketability. The identification of leaf spot caused by F. proliferatun will enable plant health authorities and farmers to identify practices to minimize disease on this important crop.
Illicium difengpi B. N. Chang et al., a shrub with aromatic odor in the Illicium genus, is extensively used as a medicinal plant in China. In June of 2020, a leaf spot on I. difengpi with incidence of about sixty percent was observed in a field located in Guilin (25°4'40"N; 110°18'21"E), Guangxi Province, China. Initial leaf symptoms were round spots with gray centers, surrounded by yellow halos. The spots gradually spread and merged. Six samples of symptomatic leaves were collected from six diseased plants, and they were surface disinfested before isolation. Potato dextrose agar (PDA) was used to culture pathogens. Successively, pure cultures were obtained by transferring hyphal tips to new PDA plates. A total of 10 isolates were obtained from the affected leaves. Two single-spore isolates (GX-1 and GX-2) were obtained and confirmed to be identical based on morphological characteristics. The representative isolate GX-2 was selected for further study on morphological and molecular characteristics. The colony of isolate GX-2 was about 4 cm in diameter on a PDA plate in 5 days, dark green with a granular surface, and irregular white edge. Conidia were hyaline, unicellular, oval, narrow at the end with a single apical appendage, and 8.2 to 13.8 × 3.7 to 7.2 µm (n = 50). Spermatia were hyaline, bacilliform with swollen ends, 3.8 to 8.9 × 1.3 to 1.9 µm (n = 50). Morphological characteristics of isolate GX-2 were consistent with the description of Phyllosticta capitalensis (Wikee et al. 2013). The internal transcribed spacer (ITS) region, translation elongation factor 1-α (tef1-α), glyceraldehyde-3-phosphate dehydrogenase (GPDH) and actin (ACT) were amplified using primers ITS1/ITS4, EF-728F/EF-986R, Gpd1-LM/Gpd2-LM and ACT-512F/ACT-783R, respectively (Wikee et al. 2013). Sequences were deposited in GenBank with accession numbers OL505439 for ITS, OL539429 for ACT, OL539430 for tef1-α and OL539431 for GPDH. BLAST analysis in GenBank showed that these sequences were 99 to 100% similar to the corresponding ITS (MT649668), ACT (MN958710), tef1-α (MN958711) and GPDH (KU716077) sequences of P. capitalensis. Also, the phylogenetic tree based on genes of ITS, tef1-α, GPDH and ACT by the maximum likelihood method showed that isolate GX-2 clustered together with P. capitalensis. The pathogenicity tests were carried out on a healthy 3 year-old plant in the greenhouse with 80% relative humidity at 25 °C. Four sterilized leaves were wounded with a needle and inoculated with 20 μL spore suspension (1 × 106 spores/ml). Another four sterilized leaves were inoculated with 20 μL sterile water as a control. All plants were incubated in a chamber with 98% relative humidity at 25 ± 1°C. After 12 days, disease symptoms similar to the field were observed on leaves, whereas control plants remained healthy. P. capitalensis was successfully reisolated only from the inoculated leaves and identified based on morphological characters. P. capitalensis caused leaf spots on various host plants around the world (Wikee et al. 2013), including on tea plants in China (Cheng et al. 2019) and oil palm in Malaysia (Nasehi et al. 2020), but it has not been reported on I. difengpi. Thus, this is the first report of P. capitalensis causing leaf spot on I. difengpi. This study will provide an important reference for the control of the disease. The epidemiology of this disease should be investigated in further research.
Cinnamomum camphora (Lauraceae), commonly known as camphor tree, is widely grown as an ornamental and is used as a source of camphor in Malaysia. In June 2021, leaves of three camphor trees with anthracnose symptoms were collected from a park (6°02'00.8"N, 116°07'18.5"E) at the Universiti Malaysia Sabah in Sabah province. The average disease severity across diseased plants was about 60% with 30% incidence on 10 surveyed plants. The disease severity on disease area of 10 leaves from each three diseased plants was estimated using ImageJ software. The disease incidence was determined based on Sharma et al. (2017). Gray spots were observed primarily on the surface of the leaves. After a week, the spots coalesced into larger patches, and anthracnose developed. Small pieces (5 x 5 mm) of symptomatic leaf tissue from three camphor trees were excised from the margin between healthy and symptomatic tissue. The pieces were surface-sterilized with 75% ethanol for 1 minute, washed with 2% sodium hypochlorite solution for 1 minute, rinsed, and air dried before plating in three Petri dishes with Potato dextrose agar, and incubated for 7 days at 25°C in the dark. After 7 days, all the PDA plates had abundant gray-white fluffy hyphae. Mycelium was dark brown when observed from the underside of the plate. The isolates UMS02, UMS04 and UMS05 were characterized morphologically and molecularly. The conidia were one-celled, cylindrical, hyaline, and smooth, with blunt ends, and ranged in size from 13.9 to 16.3 x 3.8 to 6.1 μm (n = 20). Appressoria were round to irregular in shape and dark brown in color, with size ranging from 7.8 to 9.8 μm x 5.3 to 6.8 μm (n= 20). Genomic DNA was extracted from fresh mycelium of the isolates based on Khoo et al. (2022a). Amplification of the internal transcribed spacer (ITS) region, calmodulin (CAL), actin (ACT), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes of the isolate was performed using primer pairs ITS1/ITS4, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and GDF1/GDR1 (Weir et al. 2012). PCR products with positive amplicons were sent to Apical Scientific Sdn. Bhd. for sequencing. Sequences of the isolates were deposited in GenBank as OK448747, OM501094, OM501095 (ITS), OL953034, OM513908, OM513909 (CAL), OL953031, OM513910, OM513911 (ACT), OL953037, OM513912, OM513913 (CHS-1), and OL953040, OM513914, OM513915 (GAPDH). They were 100% identical to ITS (MN296082), CAL (MN525840), ACT (MW341257, MN525819), CHS-1 (MT210318), and GAPDH (MT682399, MN525882) sequences of Colletotrichum siamense. Phylogenetic analysis using maximum likelihood on the concatenated ITS, CAL, ACT, CHS-1 and GAPDH sequences indicated that the isolates formed a clade (82% bootstrap support) to C. siamense. Morphological and molecular characterization matched the description of C. siamense (Huang et al. 2022). Koch's postulates were performed by spraying a spore suspension (106 spores/ml) on leaves of three healthy two-month-old camphor trees, while water was sprayed on three additional camphor trees which served as control. The inoculated camphor trees were covered with plastics for 48 h at 25°C in the dark, and then placed in the greenhouse. Monitoring and incubation were performed based on Chai et al. (2017) and Iftikhar et al. (2022). Symptoms similar to those observed in the field occurred 8 days post-inoculation. No symptoms occurred on controls. The experiment was repeated two more times. C. siamense has been reported causing anthracnose on camphor tree in China (Liu et al. 2022), Citrus spp. in Mexico (Pérez-Mora et al. 2021), and Crinum asiaticum and eggplant in Malaysia (Khoo et al. 2022b, 2022c). To our knowledge, this is the first report of C. siamense causing anthracnose on C. camphora in Malaysia. Our findings expand the geographic range of C. siamense and indicate it could be a potential threat limiting the camphor production of C. camphora in Malaysia.
Sansevieria trifasciata var. laurentii (De Wild.) N.E. Brown, commonly known as variegated snake plant or variegated mother-in-law's tongue, is a popular landscape and house plant. In September and October 2019, the obvious leaf spot symptoms were observed on the plants in a 0.2 hm2 of nursery in Qingdao city of China with incidence of 55%. The disease usually starts from the tip or edge of the leaf, initially have slightly water-soaked semi-circular or round brown lesions, which gradually expanded and coalesced into irregular shapes about 3-8 cm in diameter. Grayish brown sunken spots with dark margins that evolve into concentric rings of acervuli which were characteristic of anthracnose, and orange sticky conidial masses were observed under the moist condition. The leaves with typical anthracnose symptoms were collected and deposited in the herbarium of Qingdao agricultural university under accessions no. QDHB074-QDHB087. Subsequently 20 isolates with the same colony and morphological characteristics were obtained from ten diseased leaves by placing surface-sterilized tissue pieces with typical spots on potato dextrose agar (PDA). Colonies are floccose with grayish-white to dark olivaceous gray color, and gray black on the reverse after 14 days at 28°C. Straight conidia [15.0 to 27.5 × 3.5 to 7.0 μm in size (average 18.2 × 6.1 μm) (n = 50)] were cylindrical, aseptate, hyaline, slippery surface, most with one tapering end and the other oval. Setae were black, 185-230 μm in length, with a thin tip and septate in the middle. Appressoria [6.5 to 7.3 × 7.8 to 9.2 μm in size (average 6.8 × 8.1 μm) (n = 15)] were black to dark brown, solitary, spherical with smooth wall. The fungal isolates were identified as Colletotrichum sansevieriae Nakamura (Nakamura et al. 2006), based on the morphological characteristics. To confirm the identification, the internal transcribed spacer (ITS) and calmodulin (CAL) regions of a representative isolate HWL-1016 were amplified by primers ITS1/ITS4 (White et al. 1990) and CMD5/CMD6 (Weir et al. 2012), respectively. The 549 bp ITS (MN922517) and 597 bp CAL (OM994078) sequences had respectively 100% and 99.30% identity with the sequences from holotype species of C. sansevieriae MAFF 239721 (no. NR_152313 and LC180125). Phylogenetic tree based on ITS and CAL sequences respectively or jointly constructed by PAUP4.0 (Swofford 2002) revealed that the fungus in this study clustered with C. sansevieriae isolates (NR_152313, KC790947, HQ433226, JF911349, MN386823). Pathogenicity test of isolate HWL1016 was evaluated on five 3- to 4-month-old potted S. trifasciata var. laurentii under greenhouse conditions (27±2 °C, 16-hr light/8-hr dark photoperiod, 80% relative humidity). Conidial suspension (1×106 conidia/mL) of the isolated fungus from PDA colonies cultured for 15 days and sterile distilled water (as control) were sprayed on pin-pricked surface-sterilized (70% alcohol) leaves of potted plants, respectively. Three replications (three plants) were done for each treatment, and the experiment was repeated twice. The inoculated plants were covered with plastic films for 2 days and obvious water-soaked wounds were observed on the sixth day. After 16 days, the symptoms of the inoculated plants were similar to those in the nursery, with disease incidence reached 100%, while controls remained symptomless. C. sansevieriae was subsequently reisolated from the symptomatic tissues. Anthracnose on S. trifasciata var. laurentii caused by C. sansevieriae has been reported in Australia, Iran, Japan, Malaysia (Kee et al. 2020), South Korea, USA (Talhinhas & Baroncelli 2021), India (Gautam et al. 2012) and Thailand (Li et al. 2020). To our knowledge, this is the first report of C. sansevieriae causing anthracnose on S. trifasciata var. laurentii in China. This study will contribute to guide effective management based on pathogen.
Dragon fruit (Hylocereus spp.) is gaining popularity due to high net return, medicinal importance and ability to survive under less water and poor quality soils. In year 2020 and 2021, H. undatus and H. polyrhizus plants at research field of ICAR-National Institute of Abiotic Stress Management, Baramati (18°09'30.62″N, 74°30'08″E) were affected with anthracnose disease. Out of 340 plants, 60 were symptomatic, showed disease severity up to 25 to 30%. Intermittent raining in July to September and untimely rain in November, 2021 favored the disease. Infected cladodes showed reddish to dark-brown, sunken lesions, with chlorotic haloes later converted to mature necrotic patches with prominent black acervuli. On fruits, small, light brown spots quickly turned to sunken water-soaked lesions with concentric rings of black acervuli. Infected stems were collected randomly from different plants. For pathogen isolation, lesion edge tissues (5 to 10 mm2) were excised and disinfected with 1% Sodium hypochlorite (2 min) followed by triple rinsed with sterilized water and plated on potato dextrose agar (PDA) amended with Streptomycin sulphate (30 mg/L) for 4 days at 27 ± 2°C with a 12 h photoperiod. Purified colonies of three isolates 2CT, 6CT, D6CT were obtained from successive isolation attempts. Colonies were round with smooth margins, initially pale white mycelia that changed to dark gray with pinkish-orange conidial masses. Average colony diameter was 58.3 mm at 7 DAI. Conidia were single-celled, hyaline, slightly curved, tapered tip and truncate base, with an oil globule at center. Average conidia size (n=50) was 25.7 (±2.3) μm × 3.7 (±0.2) μm, L/W ratio=6.9. Conidia were initiated from an acervular conidiomata with intermittent dark brown, septate, straight, pointed setae 114 (±35) μm long × 4.5 (±1.1) μm wide. Appressoria were dark brown, lobate or round, mostly in groups, measuring 11 (±2.4) × 6.6 (±0.8) μm. Morphological characters were consistent with Colletotrichum truncatum (Schwein.) Andrus & W.D. Moore (Damm et al. 2009). For molecular identity of three isolates, partial internal transcribed spacer (ITS) region, actin, β-tubulin (TUB2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified with ITS1/ITS4, ACT512F/ACT783R, BT2A/BT2B, and GDF1/GDR1 primers, respectively. Sequences were deposited in GenBank (ITS: OK639098 to OK639100; Actin: OM927967, OM927968, ON099061; TUB2: OM927969, ON099062, ON099063; GAPDH: ON099064, ON099065). A maximum likelihood phylogenetic tree based on all sequenced loci in MEGA11 shows the clustering of present isolates in the C. truncatum clade. For pathogenicity, 4 month old unwounded stems of H. undatus and H. polyrhizus were inoculated with a spore spray (1x106/ml conidia) of C. truncatum. For each isolate, three plants were inoculated. Plants inoculated with sterilized water represented the negative control. Inoculated and control plants were kept separately at 25 ± 2°C temperature and >85% relative humidity. Inoculated plants showed minute, sunken, water soaked, reddish brown spots which were converted to sunken patches with black acervuli at 15 DAI. No symptoms were observed in the negative control. Pathogenicity test was repeated twice and the pathogen was re-isolated from symptomatic stems showed similar morphology with C. truncatum. Based on morphological and molecular characteristics and pathogenicity test, pathogen identified as C. truncatum. Previously, dragon fruit anthracnose caused by C. truncatum was reported from China (Guo et al. 2014) and Malaysia (Vijaya et al. 2015). To our knowledge, this is the first report of C. truncatum cause of dragon fruit anthracnose in India. Detailed pathogen diagnostics may help in formulating effective, on time, appropriate disease management strategies.
In July 2019 to November 2021, symptoms of fruit rot of Averrhoa bilimbi (commonly known as bilimbi) fruits were observed in Serdang (3°00'05.8"N 101°42'18.4"E) and Tanjong Karang (3°25'55.3"N 101°12'55.7"E), Selangor, Malaysia. External decay showed some yellowish to brownish-red discoloration and inside the fruit there was black powdery sporulation and a brownish decay that measured between 8 - 15 mm wide. Twenty random symptomatic fruits were collected from each location. Small pieces (5 mm) of infected tissues from the fruit rot were surface sterilized for 1 min in 0.5% NaOCl, washed twice with sterile distilled water and cultured onto potato dextrose agar (PDA) and peptone pentachloronitrobenzene agar (PPA). The plates were incubated at 28 ± 1oC under 12 hours light/dark for 7 days. The fungal colonies growing from the plates were purified using hyphal tip technique (Leyronas et al. 2012). A total of 42 fungal isolates were obtained, and the morphology characteristics of six isolates were matched that of Aspergillus niger. The A. niger isolates were further identified based genus and species-specific Internal Transcribed Spacer (ITS) sequencing. Primers ITS1/ITS4, were used to amplify and subsequently sequenced the ITS1-5.8S-ITS2 region (White et al. 1990). Primer ASAP1 (5'-CAGCGAGTACATCACCTTGG-3'), ASAP2 (5'-CCATTGTTGAAAGTTTTAACTGATT-3') was used for Aspergillus species confirmation, primer ASPU (5'-ACTACCGATTGAATGGCTCG-3') / Ni1r (5'-ACGCTTTCAGACAGTGTTCG-3') for A. niger species-specific, ASPU / Af3r (5'-CATACTTTCAGAACAGCGTTCA-3') for A. fumigatus specific-specific and ASPU / Fl2r (5'-TTCACTAGATCAGACAGAGT-3') for A. flavus specific-specific (Sugita et al. 2004). In general, Aspergillus niger isolates grew rapidly on PDA and were visibly white initially then appearing black and powdery on the second day of incubation (Figure 1A). Some isolates grew rapidly (0.71-0.85 cm/day) and have a cottony appearance. The conidia were appeared brown to black, globose and rough with diameter ranging between 4.1-5.2 µm (Figure 1B). The vesicles were hyaline, globose, and brown in color with measurement of 30-75 µm in diameter with uniseriate sterigmata (Figure 1C). The conidial head was brownish black in color and split into several irregular and regular columns of conidial chains (Figure 1D-E). The conidiophores were hyaline, and brown in color. Phylogenetic trees of ITS (Figure 2A) and ASAP sequences (Figure 2B) were constructed using a Neighbor-Joining method showing isolates Aspergillus niger #11, #15, #32, #33, #41 and #42 were grouped into the same clade as A. niger (accession no. MT446087). To examine virulence of A. niger, pathogenicity tests were performed three times by inoculating an asymptomatic fruit with six isolates of A. niger (isolate #11, #15, #32, #33, #41 and #42) and a single isolate for each species of Aspergillus aculeatus, Lasiodiplodia theobromae and Penicillium gerundense. Ten fruits were inoculated by placing a mycelial disc (6 mm) (Kouame et al. 2010) from a 5-day-old culture of each fungal colony while control fruits were non-inoculated with any fungal colony (10 fruits were inoculated with a sterile agar disc and 10 were non-inoculated, respectively). After 3 days, typical symptoms of Aspergillus fruit rot were observed on A. niger inoculated fruits, whereas the control fruits remained asymptomatic (Figure 1F-P). Aspergillus niger was reisolated and reidentified based on morphological and molecular characterization from the inoculated, symptomatic fruits, thus confirming Koch's postulates. A. niger causing widespread diseases in various plant and it is a common contaminant of food. This study shows A. niger to be highly virulent on bilimbi fruits and leads to reduction of fruit quality and its production. To our knowledge, this is the first report of A. niger causing fruit rot on bilimbi and future work on its pathogenesis may provide strategies for disease control against the pathogen.