Displaying publications 1 - 20 of 136 in total

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  1. Wei Z, Duan F, Yu D, Luo S, Yang M, Li R
    Plant Dis, 2024 Mar 18.
    PMID: 38499972 DOI: 10.1094/PDIS-09-23-1911-PDN
    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.
  2. Wang Y, Cao M, Hu T, Zhou X
    Plant Dis, 2024 Feb 06.
    PMID: 38319620 DOI: 10.1094/PDIS-12-23-2674-PDN
    Hibiscus latent Singapore virus (HLSV) and Hibiscus latent Fort Pierce virus (HLFPV) both belong to the genus Tobamovirus in the family Virgaviridae. The genomes of both HLSV and HLFPV consist of a linear positive sense single-stranded RNA of about 6.3 kb. HLSV is the causal agent of hibiscus leaf crinkle disease. Infections of HLSV in hibiscus (Hibiscus rosa-sinensis) have so far only been reported in Singapore, Japan and Malaysia (Srinivasan et al., 2002; Yoshida et al., 2018; Yusop et al., 2021). In 2017, leaf curling and chlorosis symptoms of lantana (Lantana camara) plants were found in Chenshan Botanical Garden, Shanghai, China. To detect potential virus(es) in these lantana samples, leaves from one lantana plant were collected and total RNA was extracted with RNAiso Plus (TaKaRa). A cDNA library was prepared by TruSeq RNA Sample Prep Kit (Illumina) after removing ribosomal RNA by Ribo-ZeroTM rRNA Removal Kit (Epicentre). The paired-end sequencing was then performed on an Illumina NovaSeq 6000. A total of 61,085,018 high quality reads were obtained and de novo assembly by StringTie revealed 124,516 contigs (greater than 50 bp, N50=719 bp) with an average length of 537 bp. BLASTx analyses in the National Center for Biotechnology Information (NCBI) database showed that 1 long contig of 6,305 bp, assembled of 1794 clean reads, shared significant nucleotide similarities with the genomic sequence of HLSV, and 1 contig of 6,271 bp, assembled of 3174 clean reads, shared significant similarities with the genomic sequence of HLFPV, yielding an average coverage of the whole genome at 42.65 and 75.83 per million reads, respectively. To obtain the complete genome of the viral RNA in this lantana sample, eleven overlapping regions covering the entire HLSV viral genome, and nine overlapping regions covering the entire HLFPV viral genome were amplified by reverse transcription-PCR (RT-PCR) and sequenced. In addition, the exact 5' and 3' ends of the genomic RNA of each virus were determined by rapid amplification of the cDNA ends (RACE) (Wang et al. 2020). The complete genome of the identified HLSV, deposited in GenBank: MZ020960, is 6,486 nt in length and shows 98.4% nucleotide sequence identity with HLSV Singapore isolate (GenBank: AF395898). Similar to other HLSV isolates, this virus isolate possesses an internal poly(A) tract of 87 nucleotides, which is crucial to virus replication (Niu et al., 2015). The complete genome of the Lantana HLFPV isolate is 6,463 nt (GenBank MZ020961) including a 73 nt internal poly(A) tract, and has 98.4% nt identity to HLFPV-Japan (AB917427). In two other lantana plants from the same site, the presence of HLSV and HLFPV was confirmed by RT-PCR using the primer pairs (5'-GCATCTGCATAACACGGTTG-3'/5'-ACGTTGTAGTAGACGTTGTTGTAG-3' and 5'-GGACCTTGCTAATCCGCTAAAGTTG-3'/5'-GGTCCATGTCCATCCAGATGCAATC-3'). In addition to the HLSV and HLFPV genomes, BLASTx analysis of three contigs of 3,006 bp, 2,845 bp and 2,200 bp, assembled of 1328, 352 and 2280 clean reads respectively, showed high identity to RNAs 1 (MG182148), 2 (DQ412731) and 3 (KY794710) of cucumber mosaic virus. To the best of our knowledge, this is the first report of L. camara as a new natural host of HLSV and HLFPV, and first identification of a mixed infection of HLSV and HLFPV.
  3. Palemon-Alberto F, Reyes-Garcia G, Ortega-Acosta SA, Toledo Hernandez E, Romero-Ramirez Y, Toribio-Jimenez J, et al.
    Plant Dis, 2024 Jan 24.
    PMID: 38268178 DOI: 10.1094/PDIS-09-23-1830-PDN
    Roselle (Hibiscus sabdariffa L.) is a crop of economic importance, refreshing drinks are prepared from its calyces, it is also attributed to antioxidant, antibacterial, and antihypertensive properties (Da-Costa-Rocha et al. 2014). In November 2022, in municipality of Iguala (18.355592N, 99.548546W, 749 m above sea level), Guerrero, México, roselle plants of approximately 1.5 months of age with basal rot were detected under greenhouse conditions. The symptoms consisted of wilting, yellowing, and root and stem rot with constriction in the base of the stem. The symptoms were detected in approximately 15% of plants at the operation. From symptomatic tissue, cuts were made into approximately 0.5 cm pieces, sterilized with 2% NaClO, washed with sterile distilled water, transferred to PDA medium amended with 50 mg/liter of Chloramphenicol, and incubated in the dark for four days at 28 °C. Rhizoctonia-like colonies were consistently obtained, and nine isolates were selected and purified by the hyphal-tip method. After four days, isolates developed a mycelium was light-white that became brown with age. Right-angled hyphal branching was also observed, in addition to a slight constriction at the base of the branches. In some older cultures, numerous dark brown sclerotia were observed. They were multinucleate cell with three to eight nuclei and measured from 1 to 2 mm in diameter. Together these characteristics were consistent with the description of Rhizoctonia solani Kühn (Parmeter 1970). The anastomosis group (AG) was confirmed by amplifying the ITS region with the primers ITS1 and ITS4 (White et al. 1990) of the RIJAM3 and RIJAM5 strains. The sequences were deposited in GenBank (Nos. OR364496 and OR364497 for RIJAM3 and RIJAM5, respectively). BLAST analysis, both isolates indicated 99.7 identity to R. solani AG-4 HG-I (GenBank: KM013470) strain ICMP 20043 (Ireland et al. 2015). The phylogenetic analysis of AGs sequences allowed assignment of isolates RIJAM3 and RIJAM5 to the AG-4 HG-1 clade. A pathogenicity test was performed on 20 one-month-old roselle plants. Mycelium of RIJAM3 isolate was inserted into the base of the stem with a sterile toothpick. As a control, a sterile toothpick with no mycelium was inserted in ten healthy plants. Additionally, 50 eight-day-old seedlings were inoculated by placing a 5-mm diameter agar plug colonized with mycelium of RIJAM3 at the base of the stem 10 mm below the soil surface. As control treatments, uncolonized PDA plugs were deposited at the base of 25 seedlings. The inoculated plants were incubated in a greenhouse with an average temperature and relative humidity of 28°C and 85%, respectively. Following inoculation, symptoms similar to those observed in the original outbreak were observed in plants after six days and only after four days in seedlings. In both experiments, the control plants and seedlings remained asymptomatic. R. solani was re-isolated from plants and seedlings, complying with Koch's postulates. The pathogenicity testing was repeated twice, with concordant results. In Nigeria and Malaysia R. solani was reported to seedling death to cause seedling dieback in roselle (Adeniji 1970; Eslaminejad and Zakaria 2011). In México R. solani AG-4 has been previously reported in crops of potato, chili and tomato (Montero-Tavera et al. 2013; Ortega-Acosta et al. 2022; Virgen-Calleros et al. 2000). To the best of our knowledge, this is the first report of R. solani AG-4 HG-I as a causing of root and basal stem rot on roselle in Mexico. This research provides information essential for informing the management of this disease, and may help design measures to prevent the spread of the pathogen to other regions.
  4. Afifah M, Sapak Z, Mohd Noor N, Ab Wahab MZ, Mohd Anuar IS, Ramli NW, et al.
    Plant Dis, 2024 Jan 22.
    PMID: 38254324 DOI: 10.1094/PDIS-09-23-1796-PDN
    In June 2017, severe leaf spots symptoms were observed by growers on pineapple leaves of Josapine variety in in Alor Pongsu (5°01'60.00" N, 100°34' 59.99" E), Perak, northwest of Peninsular Malaysia. The early infection stage shows that several brown spots could be observed, which then would merge to form large brown to creamy white lesions that cover all the leaf surface. This infection finally caused the plant to die after a while. Disease observations conducted from 2018 - 2023 showed that 10-15% incidences of the disease were observed in several pineapple farms located in Johor, Kedah, and Sarawak. The aim of this study to confirm the causal pathogen of the disease by performing isolation, pathogenicity testing, and identification of the primary causal pathogen from 20 samples of infected leaves collected from Alor Pongsu. The leaf tissues between infected and healthy were cut into small pieces (0.5 cm 0.5 cm), and surface sterilized with 1% sodium hypochlorite for 30 seconds, followed by 70% ethanol for 30 seconds, and rinsed thrice with sterilized water before placing on Potato Dextrose Agar (PDA). The PDA plates were incubated at room temperature (28 ± 2℃) in natural light. After five days of incubation, the potential causal pathogen was purified using a single conidial isolation technique for morphological and molecular characterizations. All 32 isolates displayed similar phenotypes. Based on morphological observation on PDA, the colonies were initially white of aerial mycelia but gradually darkened as the culture aged. Microscopic features of the 14-day-old fungal culture showed that the mycelia were branched with 0- 1 septa, pigmented, and brown. Arthroconidia were ellipsoid to ovoid or round shaped, hyaline, with rounded apex, truncate base, and occurring singly or in chains averaging 9 ± 3 × 5 ± 2 μm (n = 20).  Based on the morphological characteristics, the fungal isolates were tentatively identified as Neoscytalidium species. A representative isolate of Neoscytalidium coded as UiTMPMD2 was further identified through PCR implication of the internal transcribed spacer (ITS) region using ITS1 and ITS4 primers and BLAST homology search as Neoscytalidium dimidiatum (Penz.) Crous & Slippers based on 100% similarity (575 bp out of 575 bp) to a reference sequence (accession no. KU204558.1). The sequence was deposited in Gen Bank (accession no. OR366479) with reference sequence code of INBio:30A. Pathogenicity tests were performed on 10 whole plants of Josapine pineapple (4 months old) using a leaf inoculating method (Wu et al. 2022) in a glasshouse (25-32°C) and repeated twice. Four mature leaves per each plant were wounded at two points and inoculated with mycelium PDA plugs from 7-days-old cultures of N. dimidiatum. Control plants were wounded in the same manner but inoculated with sterilized PDA plugs. Seven days post inoculation, leaf spot symptoms were observed on treated plants with the pathogen, while the control plants remained symptomless. Pathogen was successfully reisolated from brown leaf spot symptoms in which the cultural and morphological characteristics were identical to those of the originals. Neoscytalidium dimidiatum has a wide range of hosts and it has been reported in Malaysia to cause stem canker on pitahaya (Mohd et al. 2003; Khoo et al. 2023 ) and fruit rot of guava (Ismail et al. 2021). To the best of our knowledge this is the first report of N. dimidiatum causing leaf spots on pineapples in Malaysia. This report establishes a foundation for further study of N. dimidiatum that can effectively address the disease in pineapple.
  5. Luo Z, Liu P, Qi H, Chen X, Zhang J, Sun C, et al.
    Plant Dis, 2023 Nov 30.
    PMID: 38035784 DOI: 10.1094/PDIS-03-23-0580-PDN
    Styphnolobium japonicum (L.) Schott (family Fabaceae Juss.) also called pagoda tree, is widely planted in northern China in landscape plantings, for erosion control and forestry. In recent years, symptoms of branch dieback were observed on S. japonicum in the southern part of Xinjiang province, China. From 2019 to 2022, in total ca. 1000 ha area was surveyed in Korla (41.68°N, 86.06°E), Bohu (41.95°N, 86.53°E) and Alaer (41.15°N, 80.29°E). Typical symptoms were observed in 70% of the surveyed branches. To identify the cause, we collected 50 symptomatic branches. Symptoms were initially observed on green current-year twigs, which turned grayish white in color. In the later stages of disease development, a large number of nacked black conidia formed under epidermis of perennial branches, causing visible black protrusions (pycnidia) on branch surface. The disease occurred throughout the entire growing season of S. japonicum. Symptoms also occurred on the inflorescence, fruit, and twigs. In some cases, infection resulted in tree mortality. Isolations were made from the margin between healthy and diseased tissues. Small pieces were excised, surface disinfested (75% ethanol 30 s, 1% NaClO solution 5 mins), cut into pieces (5 to 10 mm2), and incubated on PDA medium at 28℃ for 3 days. A total of 16 isolates (GH01-GH16) with similar colony morphology were obtained. The colonies were initially white, gradually turning to olive-green on the surface and black on the underside after 7 days. Microscopically, the conidia were aseptate, 1-septate, two-septate, and muriform, 2.6-4.5 × 2.9-27.6 μm (n=50). Pycnidia ranged in size from 120.2 to 135.5 × 112.4 to 118.6 µm (n=20). Those morphological characters matched the descriptions of Neoscytalidium dimidiatum (previously N. novaehollandiae) (Alizadeh et al. 2022; Pavlic et al. 2008). For molecular identification, genomic DNA of GH01-GH16 were extracted from fresh mycelia. The internal transcribed spacer (ITS), large subunit ribosomal RNA gene (LSU), and translation elongation factor 1-alpha (EF1-α) gene were amplified using the primer sets ITS1/ITS4 (White 1990), LRoR/LR5 (Vilgalys and Hester 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999). The sequences were deposited in GenBank (accession No. OP379832, OQ096643-OQ096657 for ITS, OP389048, OQ127403-OQ127417 for LSU, and OQ136617, OQ586044-OQ586058 for EF1-α). The ITS sequence had 100% identity (505/505 bp) to MT362600. Similarly, the LSU and EF1-α sequences were found to be identical to MW883823 (100%, 821/821 bp) and KX464763(99%, 256/258 bp), respectively. Pathogenicity was tested on one-year-old healthy S. japonicum seedlings. Spores of representative isolate GH01 were produced on PDA by incubating for 7-days at 28℃. Conidia were washed with sterile water. Five trees were inoculated with 1 × 106 conidia/ml conidial suspensions and five trees were sprayed with sterile water. All trees were covered with plastic bags for 24 h and kept at 25°C in a greenhouse. Signs and symptoms were similar to those observed in field collections one month after inoculation, while no symptoms occurred on the controls. The original fungus was successfully reisolated from the inoculated trees and was identified as N. dimidiatum following the methods described above. N. dimidiatum has been reported in many Asian country such as Malaysia, India, Turkey, and Iran(Akgül et al. 2019; Alizadeh et al. 2022; Khoo et al. 2023; Salunkhe et al. 2023). To our knowledge, this is the first report of N. dimidiatum associated with branch dieback of S. japonicum in China. Our findings have expanded the host range of N. dimidiatum in China and provides a theoretical basis for the diagnosis and treatment of the disease.
  6. Chang HX, Huang CC, Lu PK
    Plant Dis, 2023 Nov 21.
    PMID: 37990523 DOI: 10.1094/PDIS-10-23-2127-PDN
    From September 2020 to January 2021, an unknown disease of winged bean (Psophocarpus tetragonolobus) was reported by local growers in the Toucheng Town, Yilan County (N24.91, E121.85). The disease occurs in all age of winged bean, and the occurrence tended to be higher in humid environment, such as branches in lower canopy or branches in high density. The disease symptoms, which also appeared to be the sign of the pathogen, were spherical pustules in yellow to orange color on the stems, leaves, and pods of winged bean. Severely infected plants also exhibited growth reduction, malformation, and curling of the leaves and pods. According to the disease literature of winged bean, this unknown disease was likely to be the false rust caused by a chytrid pathogen, Synchytrium psophocarpi (UK, CAB International. 1993); and the uredinia-liked pustules could be the sori, which contain numerous ovoid to globose sporangia inside. In order to characterize the pathogen identity, the sori were manually ruptured to assess the size of individual sporangium, which had an average of 26.71 ± 4.25 μm x 26.61 ± 4.60 μm (n=42), similar to the size reported in literature (Drinkall and Price. 1979). To confirm the molecular identity, the full genomic sequences from the small subunit (SSU) to the internal transcribed spacer-1 (ITS-1), 5.8S unit, and ITS-2 were amplified using the primer sets NS3 and ITS4. The 2,263 bp amplicon was cloned and sequenced to reveal the identity (Smith et al. 2014). The BLASTN results matched the SSU of our isolate (MW649126.1) to the Synchytrium minutum (HQ324138.1) with 96% similarity (1,075 out of 1,121 bp in length), Synchytrium decipiens isolate DAOM_87618 (KF160868.1) with 92% similarity (1,215 out of 1,326 bp in length) and S. decipiens isolate AFTOL-ID 634 (DQ536475.1) with 92% similarity (1210 out of 1316 bp in length). Phylogenetic analysis using the SSU sequence revealed this unknown pathogen was the grouped within the clade of Synchytrium genus with 100% bootstrapping confidence (Smith et al. 2014). Accordingly, the pathogen was confirmed to be a Synchytrium chytrid fungus. To complete the Koch's postulates, the sori were collected from infected tissue. After vortexing washing in 1% bleach for surface sterilization, the sori were gently crashed by a plastic tube pestle to harvest sporangia. The sporangia were sprayed onto healthy winged beans cultivated in pots, and the inoculated plants were kept in a moisture bag in 25 °C. While leaf curling and malformation could be observed about 14 days post inoculation, the yellow to orange sori could be observed around 30 to 40 days post inoculation on the whole plants cultivated in pots. The sori were collected to confirm the sporangia and the sequences were identical to the original pathogen. Collectively, this study not only presents the first report for the false rust of winged bean in Taiwan, but also documents the first reference sequence of S. psophocarpi that will be useful for future molecular diagnosis. Since S. psophocarpi has been only reported in tropic regions including Indonesia, Malay Peninsula, Malaysia, Papua New Guinea, and Philippines, this report provides the first observation of S. psophocarpi moving in the subtropic region.
  7. Johari MIH, Zulperi D, Saad N, Ismail SI, Jamian S, Abdullah S, et al.
    Plant Dis, 2023 Nov 08.
    PMID: 37938907 DOI: 10.1094/PDIS-07-23-1278-PDN
    Ceylon ironwood (Mesua ferrea Linn.) or Penaga lilin is one of Asia's most popular tropical herbal plants, including Malaysia (Sharma et al., 2017). The trees are cultivated for their aesthetic value and pharmacological properties, especially as traditional remedies for asthma, dermatopathy, inflammation, and rheumatic conditions (Adib et al., 2019). In August 2022, a disease survey was conducted on Ceylon ironwood trees ranging from 5 to 12 years old in Botanical Park, Putrajaya, Malaysia, with 80% exhibiting shoot dieback disease of the 15 trees exhibiting shoot dieback disease. Symptoms include irregular, water-soaked with brown lesions on young leaves and shoots, where the small lesion coalesced and formed broad necrotic regions, subsequently causing dieback and gradual defoliation. Three infected shoots were collected from each tree, excised into small pieces (10 to 20 mm), immersed with 75% ethanol for 3 min, washed with 2% NaOCl solution for 1 min, and rinsed twice for 1 min in sterilized distilled water. A 10 µl aliquot of the sample suspension was streaked onto nutrient agar (NA) and incubated for 24 h to 48 h at 35 °C. A total of 15 isolates with similar morphology were obtained, and each isolate was re-streaked three times to obtain pure colonies that were round, smooth, with irregular edges, and produced yellow pigment in culture. All isolates were Gram-negative, negative for indole production, and utilized glucose, maltose, trehalose, sucrose, D-lactose, and pectin. Three representative isolates (C001, C002, and C003) with similar morphology were selected for further characterization. The total genomic DNA of all isolates was extracted from overnight cultures using Geneaid™ DNA Isolation Kit (Geneaid Biotech Ltd., Taiwan). PCR amplification of 16S rDNA (Zhou et al., 2015) and species-specific infB (Brady et al., 2008) genes was performed, and each of the ~1500 bp and ~900 bp amplicons were sequenced. BLASTn and phylogenetic analyses revealed all isolates were 100% identical to Pantoea anthophila (P. anthophila) LGM 2558 strains (Accession Nos. NR_116749 and NR_116113) for the 16S rDNA gene. They were 99% identical to P. anthophila CL1 strain (Accession Number CP110473) for infB gene. These sequences were later deposited in the GenBank (Accession Nos. OQ772233, OQ772234, and OQ772235 for 16S rDNA gene, and OQ803527, OQ803528, and OQ803529 for infB gene). For the pathogenicity test, healthy Ceylon ironwood seedlings' shoots were inoculated with 10 mL of each isolate suspension (1 x 108 CFU/ml) by spraying the inoculum on the young shoots using a sterilized spray bottle. Control seedlings were inoculated with sterile water. The inoculated shoots were covered with a sealed plastic bag to maintain the moisture and were kept in the greenhouse with temperatures ranging from 26 to 35 °C. The experiments were repeated twice, with three replicates for each treatment. Inoculated shoots showed dieback symptoms like natural infection, including irregular, water-soaked, and brown lesions on leaves and young shoots at 10 days post-inoculation. Control seedlings remained asymptomatic. The pathogen was re-isolated and identified via sequencing of the 16S rDNA and infB genes, thus fulfilling Koch's postulates. Previously, P. anthophila has been reported to cause soft rot in wampee plants in China (Zhou et al., 2015) and leaf blight of cotton in Pakistan (Tufail et al., 2020). To our knowledge, this is the first report of P. anthophila causing shoot dieback disease of Ceylon ironwood trees in Malaysia. Plant disease management strategies need to be established to reduce losses due to P. anthophila infection since the pathogen could limit Ceylon ironwood tree production in Malaysia.
  8. Du C, Yang D, Jiang S, Zhang J, Gao H, Ye Y, et al.
    Plant Dis, 2023 Nov 03.
    PMID: 37923973 DOI: 10.1094/PDIS-09-23-1841-PDN
    Syzygium grijsii is an evergreen shrub belonging to the family Myrtaceae, and widely cultivated in southern China as an ornamental medicinal plant. In May 2022, anthracnose symptoms were observed on leaves of S. grijsii planted in a nursery (N22°55'46″, E108°22'11″) in Nanning, Guangxi Province, China. More than 30% of leaves were infected. Initially, irregular brown spots (1 to 2 mm in diameter) formed on the leaves, with a slight depression in the center, then expanded into large, dark-brown lesions. In severe infections, lesions coalesced and covered the entire leaf, causing wilt and fall off the plant. To identify the pathogen, 30 diseased leaves were collected from five plants. Leaf tissues (5 × 5 mm) were cut from the infected margins, surface sterilized (75% ethanol 10 s, 2% NaClO 5 min, rinsed three times with sterile water), then placed on potato dextrose agar (PDA), and incubated at 28℃ in darkness. After 5 days, 16 fungal isolates with similar morphology were obtained from 30 plated tissues. Colonies on PDA were abundant with grayish-white fluffy mycelia, and yellowish-white on the back. Conidia were one-celled, hyaline, smooth-walled, cylindrical with narrowing at the center, blunt at the ends, and ranged from 11.35 to 22.14 × 4.88 to 7.67 μm (n=100). Morphological characteristics of the isolates were similar to the descriptions of Colletotrichum sp. (Prihastuti et al. 2009). Five representative isolates (Cs34, Cs31, Cs32, Cs33 and Cs35), which were preserved in the Guangxi Key Laboratory of Biology for Crop Diseases and Insect Pests, were selected for molecular identification. The ITS (Nos. OQ618199, OR539576 to OR539579), TUB2 (Nos. OQ630972, OR545076 to OR545079), ACT (Nos. OQ685919, OR545060 to OR545063), CHS-1 (Nos. OQ685917, OR545068 to OR545071), GAPDH (Nos. OQ685916, OR545072 to OR545075), and CAL (Nos. OQ685918, OR545064 to OR545067) sequences showed >99% identity to those of Colletotrichum siamense ex-type culture ICPM 18578 (Nos. JX010171, JX009924, JX009714 and JX009518) and strain C1315.2 (Nos. JX009865 and JX010404) in GenBank. Multigene phylogenetic analyses (ITS, TUB, ACT, CHS-1, GAPDH, and CAL) using the Maximum likelihood method indicated that the 5 isolates were clustered with C. siamense. To perform pathogenicity tests, three one-year-old healthy S. grijsii plants were inoculated with conidial suspension (1 × 106 conidia/ml) of isolate Cs34 by brushing gently with a soft paintbrush, each plant was inoculated with 3 leaves. The same number of plants were inoculated with sterile water as control, and pathogenicity tests were performed three times. All plants were kept in an artificial climatic box at 28℃, with a 90% humidity and a 12 h light/dark cycle. Similar symptoms to those of the field were observed on all inoculated leaves after 5 days, whereas controls remained symptomless. Reisolated fungi from the diseased leaves were confirmed to be C. siamense by morphology and molecular characterization, confirming Koch's postulates. C. siamense has been reported causing anthracnose on Crinum asiaticum (Khoo et al. 2022) in Malaysia, and Erythrina crista-galli in China (Li et al. 2021). To our knowledge, this is the first report of C. siamense causing anthracnose on S. grijsii in China. The results of pathogen identification provide crucial information for control strategies of the disease.
  9. Inokuti EM, Saraiva JLR, Silva DEMD, Corrêa MCM, Lima CS
    Plant Dis, 2023 Oct 26.
    PMID: 37884480 DOI: 10.1094/PDIS-09-23-1985-PDN
    In November 2021, stem gray blight symptoms were seen on two dragon fruit (pitaya) species (Hylocereus megalanthus and H. polyrhizus) in an orchard with 100% disease incidence in Fortaleza, Ceará, Brazil (3°44'24.5"S 38°34'30.8"W). The symptoms were initially yellowish to dark brown lesions, and as the symptoms progressed, the lesions turned grayish with small black pycnidia in the center. Isolation was carried out by disinfecting small pieces of the symptomatic stems in 70% ethanol for 1 min, followed by 1% NaOCl for 1 min, and then rinsed three times with sterile distilled water. Excess water was removed using sterile filter paper. Then the stem fragments were placed on PDA media. Colonies produced small black pycnidia with conidia and some were sterile after 68 days of incubation. Two monosporic isolates were obtained from the colonies: UFCM 0708 from H. megalanthus and the UFCM 0710 from H. polyrhizus, which were used for pathogenicity test, morphological and molecular identification. The colony on PDA was smoke gray with aerial mycelium and the reverse was smoke grey to dark grey. The α-conidia from UFCM 0708 and UFCM 0710 were hyaline, aseptate and fusiform and measured 6.4 to 9.7 (8.0) x 1.2 to 2.4 (1.7) µm and 6 to 13.1 (8.2) x 1.7 to 2.4 (2.0) µm, respectively. The β-conidia from UFCM 0708 and UFCM 0710 were hyaline, aseptate and filiform and measured 15 to 22.5 (18.8) x 0.6 to 1.7 (1.0) µm, and 17.2 to 27.5 (22.3) x 0.5 to 1.0 (0.8) µm (n=30), respectively. This morphology placed the isolates as Diaporthe sp. (Udayanga et al. 2012). For further confirmation, genomic DNA was extracted from the isolates (UFCM 0708 and UFCM 0710), and beta-tubulin (TUB2) and translation elongation factor 1-alpha (TEF1) gene fragments were amplified. BLASTn search results with isolates TEF1 and TUB2 sequences varied from 98.58% to 99.52% identity to the ex-type sequence of Diaporthe arecae (CBS 161.64). Phylogenetic analysis of concatenated sequences alignment carried out using the Maxinum-likelihood and Bayesian Inference analysis placed the isolates within D. arecae clade with 86% bootstrap and 0.99 posterior probabilities support. The sequences obtained in this study were deposited in GenBank (TEF1: OP534720 and OP534722; TUB2: OP534717 and OP534719). The isolates were confirmed as D. arecae based on molecular analysis and morphological characteristics (Gomes et al. 2013). Koch's postulates were completed as described by Karim et al. (2019) through the inoculation of six stems of each dragon fruit (pitaya) species. The stems were wounded by removing a 5 mm diameter disc and after that they were inoculated with a 5 mm diameter mycelial plug from 5 days old PDA plates. PDA plugs were used as control. Each stem was covered with a plastic bag and sterilized water was added into the sterilized filter paper to maintain humidity. The bags were kept in a room at day and night temperature of 25 ± 2 °C. The same symptoms seen in the field appeared on the stems 21 days after inoculation. The control stems remained symptomless. Diaporthe arecae have been reported on H. polyrhizus in Malaysia (Huda-Shakirah et al. 2021). To our knowledge, this is the first report of D. arecae on H. megalanthus and H. polyrhizus in Brazil.
  10. Ishak NF, Wan Azhar WMA, Ahmad S, Khairuddin AU, Laboh R
    Plant Dis, 2023 Oct 19.
    PMID: 37858968 DOI: 10.1094/PDIS-06-23-1076-PDN
    In Malaysia, bell pepper (Capsicum annuum var. grossum), also known as sweet pepper or paprika, is one of the highly imported vegetable crops. In 2021 alone, Malaysia imported nearly 74 thousand metric tons of its chilies, including bell peppers, from other countries (DOSM, 2022). Often, farmers grow the bell peppers in moderate to cool conditions within highland regions for local commercial purposes. In June 2022, the Malaysian Agricultural Research and Development Institute (MARDI) in Serdang, Selangor, conducted a research study to grow lowland bell peppers under a glasshouse rain protection system. A disease inspection carried out found fruit rot on approximately 30% of mature bell pepper fruits in the greenhouse. Symptoms appeared as firm and sunken black lesions covered with white to light pink spore masses on the outer surface, which eventually fell off. Infected fruit parts were disinfected with 10% hypochlorite (NaOCl) for 2 min, followed by double washing with sterile distilled water, air-dried, and placed onto potato dextrose agar (PDA). After 3 days of incubation, the fungal colonies that grew from the symptomatic tissue pieces were transferred onto new PDA to obtain pure cultures. The pure fungal colony appeared dense, whitish aerial mycelium that slowly became cream to pinkish-orange after 7 days of incubation at room temperature (25±2 °C). To examine the morphology features, the pure cultures were subbed onto carnation leaf agar (CLA) and incubated at 25±2°C for 14 days. Macroconidia were abundant, slightly curved with tapered apical cells, 3- to 5-septate, and ranged between 21.8 and 34.0 x 3.0 and 5.1 μm. Microconidia were single-celled, often 1-septate, and ranged between 10.0 and 12.6 x 2.1 and 3.4 μm. Chlamydospores were globose and in chains. The fungus was identified as Fusarium sp. according to Fusarium key by Leslie and Summerell (2006). PCR amplification and DNA sequencing were performed using primers EF1F/EF2R and ITS1/ITS4 (O'Donnell et al., 1998; White et al., 1990) to amplify the partial elongation factor 1-alpha (TEF1-α) gene and internal transcribed spacer region (ITS), respectively. The TEF1-α and ITS sequences of this isolate were deposited in GenBank as OQ672911 and OR349657. BLAST analysis with TEF1-α gene sequences revealed 99.74% and 99.33% sequence identity with F. pernambucanum (accession no. ON330424) and Fusarium isolate NRRL 25134 (accession no. JF740755), respectively; both belonged to the Fusarium incarnatum-equiseti species complex (FIESC). BLAST search of the TEF1-α sequence in the database of the International Mycological Association (www.mycobank.org) showed 99.18% identity with FIESC (NRRL 36548). The ITS sequences were 100% identical to those of F. incarnatum (MT563420, MT563419, and MT563418). Pathogenicity test was conducted on three unwounded and three wounded mature red bell pepper fruits (SP299 Red Masta variety). Two healthy bell peppers were used as controls for each treatment. Prior to inoculation, the fruits were surface-sterilized by dipping in 70% ethanol and rinsed twice with sterile distilled water. Unwounded fruits were inoculated with fungal mycelium disks (5 mm diameter), whereas control fruits were inoculated with sterile PDA agar disks. For wound method, 6 µl of spore suspension (1x106 spores/ml) was obtained from 7-day-old cultures and injected (1 mm depth) into the fruit wall using a sterile syringe needle. Control fruits were inoculated with sterile distilled water only. Each fruit was inoculated with the inoculum at three distinct spots and kept in a humid chamber at a temperature of 25±2 °C. The pathogenicity test was done twice. Five days post-inoculation, the control fruits showed no symptoms, whereas all inoculated wounded and non-wounded fruits developed necrotic lesions with white mycelium growing on the inoculation points. The pathogen was successfully re-isolated from the infected fruits and morphologically identified as FIESC, fulfilling Kochs postulates. It has been reported previously that the members of FIESC are responsible for the fruit rot of bell peppers under greenhouse conditions (Ramdial et al., 2016). To the best of our knowledge, this is the first report of FIESC causing fruit rot on greenhouse bell peppers in Malaysia. This fruit rot disease may impose significant constraints on bell pepper production in Malaysia; hence, effective strategies to control the pathogen and prevent disease dispersal should be implemented.
  11. Márquez-Licona G, García-León E, Flores-Moctezuma HE, Solano-Báez AR
    Plant Dis, 2023 Sep 27.
    PMID: 37755414 DOI: 10.1094/PDIS-04-23-0797-PDN
    Frangipani (Plumeria rubra L.; Apocynaceae.) is a deciduous ornamental shrub, native to tropical America and widely distributed in tropical and subtropical regions. In Mexico, P. rubra is also used in traditional medicine and religious ceremonies. In November 2018-2022, rust-diseased leaves of P. rubra were found in Yautepec (18°49'29"N; 99°05'46"W), Morelos, Mexico. Symptoms of the disease included small chlorotic spots on the adaxial surface of the infected leaves, which as the disease progressed turned into necrotic areas surrounded by a chlorotic halo. The chlorotic spots observed on the adaxial leaf surface coincided with numerous erumpent uredinia of bright orange color on the abaxial leaf surface. As a result of the infection, foliar necrosis and leaves abscission was observed. Of the 40 sampled trees, 95% showed symptoms of the disease. On microscopic examination of the fungus, bright orange, subepidermal uredinia were observed, which subsequently faded to white. Urediniospores were bright yellow-orange color. They were ellipsoid or globose, sometimes angular, echinulate, (21.5) 26.5 (33.0) × (16.0) 19.0 (23.0) μm in size. Morphological features of the fungus correspond with previous descriptions of Coleosporium plumeriae by Holcomb and Aime (2010) and Soares et al., (2019). A voucher specimen was deposited in the Herbarium of the Departmet of Plant-Insect Interactions at the Biotic Products Development Center of the National Polytechnic Institute under accession no. IPN 10.0113. Species identity was confirmed by amplifying the 5.8S subunit, the ITS 2 region, and part of the 28S region with rust-specific primer Rust2inv (Aime, 2006) and LR6 (Vilgalys and Hester 1990). The sequence was deposited in GenBank (OQ518406) and showed 100% sequence homology (1435/1477bp) with a reference sequence (MG907225) of C. plumeriae from Plumeria spp. (Aime et al. 2018). Pathogenicity was confirmed by spraying a urediniospores suspension of 2×104 spores ml-1 onto ten plants of P. rubra. Six plants were inoculated and sealed in plastic bags, while four noninoculated plants were applied with sterile distilled water. Plants were inoculated at 25°C and held for 48 h in a dew chamber, after this, the plants were transferred to greenhouse conditions (33/span>2°C). The experiment was performed twice. All inoculated plants developed rust symptoms after 14 days, whereas the non-inoculated plants remained symptomless. The recovered fungus was morphologically identical to that observed in the original diseased plants, thus fulfilling Koch's postulates. According to international databases (Crous 2004; Farr and Rossman 2023), C. plumeriae has not been officially reported in Mexico, despite being a prevalent disease. Diseased plants have been collected and deposited in herbaria, unfortunately, these reports lack important information such as geographic location of sampling, pathogenicity tests, or molecular evidence, which are essential for a comprehensive study of the disease in Mexico. To our knowledge, this is the molecular confirmation of Coleosporium plumeriae causing rust of Plumeria rubra in Mexico. Rust of P. rubra caused by C. plumeriae has been previously identified in India, Taiwan, Malaysia, and Indonesia by Baiswar et al. (2008), Chung et al. (2006), Holcomb and Aime (2010) and Soares et al., (2019). This disease causes important economic losses in nurseries, due to the defoliation of infected plants.
  12. Chong D, Alsultan W, Ariff SNH, Kong LL, Ho CL, Wong MY
    Plant Dis, 2023 Sep 14.
    PMID: 37709725 DOI: 10.1094/PDIS-04-23-0636-PDN
    Coconut (Cocos nucifera) is a high economic value cash crop in Malaysia. In December 2021, irregular spots with dotted rust-like appearance were observed mainly on the tip of the leaves of MATAG variety coconut seedlings at the nursery in Perak state. More than 90% of the coconut seedlings surveyed were infected with leaf spot symptoms. These symptoms could bring huge economic losses due to the downgrade value of the seedlings. 15 symptomatic leaves were obtained from the nursery, 10 mm2 of cut leaves were disinfected with 10% sodium hypochlorite for 10 minutes and rinsed with sterile distilled water before plated on potato dextrose agar (PDA). A total of 4 single-spore isolates were obtained and were observed morphologically. The isolates had white cotton-like appearance with undulate edge. Black acervuli were seen after 7 days of incubation at 26 °C. The conidia were fusiform and contained five cells with four septate and three versicolor cells in between the apical and basal cell. The conidia were 17.2 µm long and 5.9 µm wide (n=30). Conidia consisted of two to three apical appendages and one basal appendage. These morphological characters were consistent with the original description of Neopestalotiopsis clavispora (Santos et al., 2019; Abbas et al., 2022). Species identification was done by amplifying internal transcribed spacer (ITS) region using primers ITS 4 and ITS 5 (White et al., 1990) and beta-tubulin (TUB2) using primers Bt2a and Bt2b (Glass & Donaldson et al., 1995) of the representative isolate LKR1, then sequenced. The 488 bp ITS and 409 bp TUB2 sequences were deposited in GenBank under the accession numbers ON844193 and OP004810, respectively. Isolate LKR1 shares 99.8% identity with the ITS sequence (MH860736.1) of the reference pathogenic N. clavispora strain CBS:447.73 and 100% identity with the TUB2 sequence (KM199443.1) of the reference pathogenic N. clavispora strain CBS 447.73. The phylogenetic analysis confirmed that the isolate LKR1 belonged to N. clavispora when a supported clade is formed with 98% and 94% bootstrap support for ITS and TUB2 respectively with other related N. clavispora. Pathogenicity test was conducted by using five replicates of 8 month old seedlings, they were incubated under greenhouse condition and were watered daily. The leaves of the seedlings were injured with sterile needles and were sprayed with conidial suspension (1 x 10^6 conidia/ml). The control plants were also injured but sprayed with sterile distilled water. After a month, signature symptoms of spots on the leaves appear but none on the control seedling. N. clavispora was successfully re-isolated only from the inoculated symptomatic leaves and identified morphologically. No fungus was re-isolated from the control seedlings. The result was consistent even after repeating the test one more time. N. clavispora has been reported causing leaf spot on Macadamia integrifolia (Santos et al., 2019), Phoenix dactylifera L. (Basavand et al., 2020) and Musa acuminata (Qi et al., 2022). N. clavispora has also been reported causing rust-like appearance of leaves on strawberry (Fragaria × ananassa Duch.) (Obregón et al., 2018). To our knowledge, this is the first report of N. clavispora causing leaf spot disease on coconut seedlings in Malaysia. Through the identification of N. clavispora as the causal agent of leaf spot on coconut, this can help coconut growers to tackle the disease problem earlier thus, preventing the disease from spreading until the adult phase.
  13. Deepak Reddy B, Srilatha P, Murthy KGK, Madhusudhan Reddy S, Reddy IVS, Neelima P, et al.
    Plant Dis, 2023 Sep 08.
    PMID: 37682227 DOI: 10.1094/PDIS-08-23-1563-PDN
    Averrhoa carambola (Star fruit) is a drought resistant edible fruit belongs to family Oxalidaceae. It is native of Malaysia and further cultivation is extended to China, Southeast Asia, India and Northern South America. Star fruit has juicy texture and used in salads, beverages and traditionally it has been used for ayurvedic medicines in India, Brazil and China (Abduh et al. 2023). In early January 2023, we observed the symptoms of raised, more or less circular, orange to dark brown, velvet textured, scattered algal leaf spots (1-4 mm) on the upper surface of A. carambola leaves at College farm, Agricultural College, Aswaraopet (17.252039 latitude, 81.109573 longitude) (Supplementary Fig 1). The disease was observed in 2 hectare model orchard with incidence of 45% causing leaf defoliation and thereby reducing the yield and quality of fruits. Transverse section cutting of algal spots revealed the algal thalli at subcuticular region and causing necrosis of epidermal cells. Sporangiophores (n=20) raised from algal leaf spot were cylindrical, 4 to 5 celled, 200-450 µm long x 8-20 µm wide, and forming a head cell with suffultory cells and sporangia on the top. Sporangia (n=20) were spherical to elliptical, rusty brown and 17.5-29 µm long × 18-23.6 µm wide and the total number of sporangia produced by each sporangiophores varies from 1 to 6. Setae (n=20) were filamentous with three to six celled, 17.5-50 µm long × 2.5-7.5 µm wide (Supplementary Figure 2). In our collection, mature gametangia were not observed. Morphological characters were studied on 20 diseased leaf samples collected from randomly selected five plants. To isolate pathogen, fresh algal thalli (n=5) were scraped from host tissue, surface sterilized (70% alcohol (30 s), 1% sodium hypochlorite (30 s) and sterile distilled water (3 × 60 s), inoculated to trebouxia liquid media and incubated at 25 ± 2 °C with a 12 hours photoperiod for 72 hours (Vasconcelos et al. 2018). The resultant five algal filaments were subjected to PCR amplification. The primer pair PNS1/NS41 was used in a PCR to amplify a fragment of 18S rRNA (Davis and Kaur 2019). The 18S rRNA gene sequences of the algae were compared using the Basic Local Alignment Search Tool (BLAST; http://www.ncbi.nlm.nih.gov/Blast/Blast.cgi) showed that our partial sequence had 99.5% similarity to C. virescens (KM020142.1). Hence, it was classified as C. virescens and sequences was deposited in NCBI-GenBank with accession numbers (OR053653, OR243777, OR429406, OR429407 and OR243779). For proving pathogenicity, algal filaments obtained from trebouxia liquid media were inoculated to 6 months old healthy A. carambola plant. Pathogenicity test was negative and typical symptoms could not be produced even up to 150 days of inoculation. In previous studies also, due to difficulty with production of zoospores in synthetic media, Koch's postulates of C. virescens as a plant pathogen has not been demonstrated experimentally (Sunpapao et al. 2017; Sanahuja et al. 2018; Kumar et al. 2019). In the second experiment, zoosporangia spore suspension were prepared from small pieces of algal leaf spot tissue processed in a sterile pestle and mortar and filtered through sterile cheesecloth (Sunpapao et al. 2017). A total of five isolates of zoosporangia spore suspension (1 x 102 to 1 x 104/ml of water) was sprayed on healthy, surface sterilized leaves of A. carambola plants (n=5) until runoff with a handheld airpump sprayer and incubated in green house (T: 25 oC, H: 80%). During the experiment leaves were remain attached to plant (5 days old) and plants were 6 months old grown in plastic pots under controlled conditions. Two plants were inoculated with each isolate and three non inoculated control plants were included. Non inoculated controls were sprayed with sterile distilled water. The pathogenicity experiment was repeated. The initial symptoms were produced 60 days after inoculation and complete algal thalli was observed on 90 days after inoculation, control plants were without any symptoms upto 150 days. Reisolated algal thalli from symptomatic plants were morphologically similar to original algal thalli and molecularly identified as C. virescens (accession number OR067193 and OR243810). Red rust caused by C. virescens is a major algal disease in the world and causing severe leaf defoliation in various horticultural crops viz., Mangifera indica (Vasconcelos et al. 2018), Manilkara zapota (Sunpapao et al. 2017), Psidium guajava (Rajbongshi et al. 2022), Ziziphus mauritiana (Shareefa et al. 2022) and Anacardium occidentale (Dooh et al. 2022). The available literature suggest that, this is the first report of algal leaf spot on A. carambola caused by C. virescens in India. This report extends the range of known pathogens associated with A. carambola plant and serves as a basis for development and implementing disease management strategies.
  14. Teoh SH, Wong GR, Teo WFA, Mazumdar P
    Plant Dis, 2023 Aug 03.
    PMID: 37537794 DOI: 10.1094/PDIS-06-23-1239-PDN
    Brassica rapa var. Chinensis (curly dwarf pak choy) is commonly grown in large-scale vertical farming aquaponic systems. In October 2022, soft rot symptoms and dark brown lesions were observed on B. rapa grown in a commercial aquaponic farm located in Perak, Malaysia. The infected stem appeared brown and water soaked. Severely infected plants produced creamy white ooze on the surface before collapsing entirely (Fig. 1A and B). Infected leaves displayed yellow-brown symptoms and eventually rotted (Fig. 1C); the healthy plants were symptomless (Fig. 1D). About 20 % of the 20,000 B. rapa plants on the farm exhibited symptoms. Ten randomly selected symptomatic plants, five with infected stems and five with infected leaves, were surface sterilized. Each tissue (1.0 cm2) was homogenized and suspended in a saline solution. The suspensions were then serially diluted and plated separately on Luria-Bertani agar. After a 16-h incubation period, stem tissue yielded 12 isolated colonies, while leaf tissue produced 8 colonies. These isolates were subjected to dereplication using RAPD-PCR (Krzewinski et al., 2001), revealing two distinct RAPD patterns. The cultures, named Pathogen Stem 2 (PS2, obtained from the stem) and Pathogen Leaf 2 (PL2, obtained from the leaf), were initially identified as Pectobacterium sp. through 16S rRNA sequence analysis (Frank et al., 2008) on the EzBioCloud 16S database (Yoon et al., 2017). Further identification of the Pectobacterium species was conducted using multilocus sequence analysis (MLSA) of the icdA, mdh, proA, and mltD genes (Ma et al., 2007). The sequences were deposited in GenBank (OQ660180, OQ660181, and OR206482-OR206489). Based on MLSA phylogeny, PS2 and PL2 were identified as Pectobacterium carotovorum and Pectobacterium aroidearum, respectively (Fig. 2A). Anaerobic assays confirmed their facultative anaerobic nature, while Gram staining revealed Gram-negative, rod-shaped morphology consistent with Pectobacterium (Fig. 2B and C). For the re-inoculation study, one-month-old healthy B. rapa plants were used. PS2 was inoculated into petioles, while PL2 was inoculated into leaves separately (3 biological replicates × 3 leaves for each replicate) using the prick inoculation method (Wei et al., 2019). Sterile needles were used to prick the plant tissues, and 10 µL of bacterial suspensions (2.40×109 CFU/mL) in saline were inoculated onto the pricked spots. Negative control using sterile saline was included. The inoculated plants were maintained in a controlled growth chamber (25 ± 1°C, relative humidity 80 ± 5%). After 48 hpi, the petiole tissue inoculated with PS2 showed bacterial soft rot symptoms (Fig. 1F) and leaves inoculated with PL2 appeared dark brown around the wound (Fig. 1G), similar to the symptoms observed in the commercial farm (Fig. 1B, C); while control plants remained asymptomatic (Fig. 1E). Bacteria were re-isolated from the inoculated petiole and leaf tissue and their identities were confirmed by RAPD-PCR. The RAPD profiles of the bacteria reisolated from the petiole and leaf tissues were the same as those of PS2 and PL2 respectively (Fig. 1H). The pathogenicity of PS2 and PL2 was thus confirmed. To our knowledge, this is the first report of bacterial soft rot on B. rapa in aquaponic systems caused by P. carotovorum and P. aroidearum in Malaysia. The identification of these pathogens is crucial for the prevention of disease outbreaks and to develop an effective disease management strategy.
  15. Liao TZ, Chen YH, Tsai JN, Chao C, Huang TP, Hong CF, et al.
    Plant Dis, 2023 Jul;107(7):2039-2053.
    PMID: 36428260 DOI: 10.1094/PDIS-06-22-1285-RE
    Brown root rot disease (BRRD), caused by Phellinus noxius, is an important tree disease in tropical and subtropical areas. To improve chemical control of BRRD and deter emergence of fungicide resistance in P. noxius, this study investigated control efficacies and systemic activities of fungicides with different modes of action. Fourteen fungicides with 11 different modes of action were tested for inhibitory effects in vitro on 39 P. noxius isolates from Taiwan, Hong Kong, Malaysia, Australia, and Pacific Islands. Cyproconazole, epoxiconazole, and tebuconazole (Fungicide Resistance Action Committee [FRAC] 3, target-site G1) inhibited colony growth of P. noxius by 99.9 to 100% at 10 ppm and 97.7 to 99.8% at 1 ppm. The other effective fungicide was cyprodinil + fludioxonil (FRAC 9 + 12, target-site D1 + E2), which showed growth inhibition of 96.9% at 10 ppm and 88.6% at 1 ppm. Acropetal translocation of six selected fungicides was evaluated in bishop wood (Bischofia javanica) seedlings by immersion of the root tips in each fungicide at 100 ppm, followed by liquid or gas chromatography tandem mass spectrometry analyses of consecutive segments of root, stem, and leaf tissues at 7 and 21 days posttreatment. Bidirectional translocation of the fungicides was also evaluated by stem injection of fungicide stock solutions. Cyproconazole and tebuconazole were the most readily absorbed by roots and efficiently transported acropetally. Greenhouse experiments suggested that cyproconazole, tebuconazole, and epoxiconazole have a slightly higher potential for controlling BRRD than mepronil, prochloraz, and cyprodinil + fludioxonil. Because all tested fungicides lacked basipetal translocation, soil drenching should be considered instead of trunk injection for their use in BRRD control.
  16. Mejías Herrera R, Hernández Y, Magdama F, Mostert D, Bothma S, Paredes Salgado EM, et al.
    Plant Dis, 2023 Jun 20.
    PMID: 37340554 DOI: 10.1094/PDIS-04-23-0781-PDN
    Fusarium wilt of banana (Musa spp.), caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc), is a major constraint to banana production worldwide (Dita et al., 2018). A strain of Foc that affects Cavendish (AAA) bananas in the tropics, called Foc tropical race 4 (TR4; VCG 01213), is of particular concern. Foc TR4 was first detected in Malaysia and Indonesia around 1990 but was restricted to Southeast Asia and northern Australia until 2012. The fungus has since been reported from Africa, the Indian subcontinent, and the Middle East (Viljoen et al., 2020). Foc TR4 was detected in Colombia in 2019 and in Perú in 2021 (Reyes-Herrera et al., 2020). The incursions into Latin America and the Caribbean (LAC) triggered global concerns, as 75% of international export bananas are produced in the region. Banana production in Venezuela, however, is primarily intended for domestic consumption (Aular and Casares, 2011). In 2021 the country produced 533,190 metric tons of banana on an area of 35,896 ha, with an approximate yield of 14,853 kg/ha (FAOSTAT, 2023). In July 2022, severe leaf-yellowing, and wilting, along with internal vascular discoloration of the pseudostem, were noted in Cavendish banana plants cultivar 'Valery' in the states of Aragua (10°11'8″N; 67°34'51″W), Carabobo (10º14'24″N; 67º48'51″W), and Cojedes (9°37'44″N; 68°55'4″W). Necrotic strands from the pseudostems of diseased plants were collected for identification of the causal agent using DNA-based techniques, vegetative compatibility group (VCG) analysis and pathogenicity testing. The samples were first surface disinfected and plated onto potato dextrose agar medium. Single-spored isolates were identified as F. oxysporum based on cultural and morphological characteristics, including white colonies with purple centres, infrequent macroconidia, abundant microconidia on short monophialides, and terminal or intercalary chlamydospores (Leslie and Summerell, 2006). Foc TR4 was identified from five isolates by endpoint and quantitative-PCR using four different primer sets (Li et al. 2013; Dita et al. 2010; Aguayo et al. 2017; Matthews et al. 2020). The same isolates were identified as VCG 01213 by successfully pairing nitrate non-utilizing (nit-1) mutants of the unknown strains with Nit-M testers of Foc TR4 available at Stellenbosch University (Leslie and Summerell, 2006). For pathogenicity testing, 3-month-old Cavendish banana plants cultivar 'Williams' were inoculated with isolates from Venezuela grown on sterile millet seed (Viljoen et al., 2017). Plants developed typical Fusarium wilt symptoms 60 days after inoculation, including yellowing of leaves that progressed from the older to the younger leaves, wilting, and internal discoloration of the pseudostem. Koch's postulates were fulfilled by reisolating and identifying Foc TR4 from the plants by qPCR (Matthews et al., 2020). These results provide scientific proof of the presence of Foc TR4 in Venezuela. The Venezuelan Plant Protection Organization (INSAI) has declared Foc TR4 as a newly introduced pest (January 19, 2023), and infested banana fields were placed under quarantine. Comprehensive surveys are now conducted in all production areas in Venezuela to assess the presence and impact of Foc TR4, and information campaigns were started to make farmers aware of biosecurity protocols. Collaborative initiatives and coordinated actions among all stakeholders are needed to prevent the spread of Foc TR4 to other countries in Latin America, and to develop Foc TR4-resistant bananas (Figueiredo et al. 2023).
  17. Khoo YW, Gao L, Khaw YS, Tan HT, Li S, Chong KP
    Plant Dis, 2023 May 25.
    PMID: 37227434 DOI: 10.1094/PDIS-01-23-0109-PDN
    Paspalum conjugatum (family Poaceae), locally known as Buffalo grass, is a perennial weed that can be found in rice field, residential lawn, and sod farm in Malaysia (Uddin et al. 2010; Hakim et al. 2013). In September 2022, Buffalo grass with rust symptoms and signs were collected from the lawn located in Universiti Malaysia Sabah in the province of Sabah (6°01'55.6"N, 116°07'15.7"E). The incidence was 90%. Yellow uredinia were observed primarily on the abaxial surface of the leaves. As the disease progressed, leaves were covered with coalescing pustules. Microscopic examination of pustules revealed the presence of urediniospores. Urediniospores were ellipsoid to obovoid in shape, contents in yellow, 16.4-28.8 x 14.0-22.4 μm and echinulate, with a prominent tonsure on most of the spores. A fine brush was used to collect yellow urediniospores, and genomic DNA was extracted based on Khoo et al. (2022a). The primers Rust28SF/LR5 (Vilgalys and Hester 1990; Aime et al. 2018) and CO3_F1/CO3_R1 (Vialle et al. 2009) were used to amplify partial 28S ribosomal RNA (28S) and cytochrome c oxidase III (COX3) gene fragments following the protocols of Khoo et al. (2022b). The sequences were deposited in GenBank under accession numbers OQ186624- OQ186626 (985/985 bp) (28S) and OQ200381-OQ200383 (556/556 bp) (COX3). They were 100% similar to Angiopsora paspalicola 28S (MW049243) and COX3 (MW036496) sequences. Phylogenetic analysis using maximum likelihood based on the combined 28S and COX3 sequences indicated that the isolate formed a supported clade to A. paspalicola. Koch's postulates were performed with spray inoculations of urediniospores suspended in water (106 spores/ml) on leaves of three healthy Buffalo grass leaves, while water was sprayed on three additional Buffalo grass leaves which served as control. The inoculated Buffalo grass were placed in the greenhouse. Symptoms and signs similar to those of the field collection occurred after 12 days post inoculation. No symptoms occurred on controls. To our knowledge, this is the first report of A. paspalicola causing leaf rust on P. conjugatum in Malaysia. Our findings expand the geographic range of A. paspalicola in Malaysia. Albeit P. conjugatum is a host of the pathogen, but the host range of the pathogen especially in Poaceae economic crops need to be studied. Weed management could be an effective way to eliminate inoculum sources of A. paspalicola.
  18. Yang X, Colburn C, Roach K, Zee T, Long S
    Plant Dis, 2023 May 23.
    PMID: 37221241 DOI: 10.1094/PDIS-04-23-0701-PDN
    In February 2023, two Monstera deliciosa Liebm. (Araceae) plants with typical symptoms of leaf rust disease were detected at a grocery store in Oconee Co., South Carolina. Symptoms included chlorotic leaf spots and abundant brownish uredinia, mainly on the adaxial surface of more than 50% of leaves. The same disease was detected on 11 out of 481 M. deliciosa plants in a greenhouse at a plant nursery located in York Co., South Carolina, in March 2023. The first plant sample detected in February was used for morphological characterization, molecular identification, and pathogenicity confirmation of the rust fungus. Urediniospores were densely aggregated, globose, golden to golden brown in color, and measured 22.9 to 27.9 µm (aver. 26.0 ± 1.1 µm; n=50) in diameter with wall thickness at 1.3 to 2.6 µm (aver. 1.8 ± 0.3 µm; n=50). Telia were not observed. These morphological traits aligned with those of Pseudocerradoa paullula (basionym: Puccinia paullula; Ebinghaus et al. 2022; Sakamoto et al. 2023; Sydow and Sydow 1913; Urbina et al. 2023). Genomic DNA was extracted from urediniospores collected from the naturally infected plant sample and used for PCR amplification and DNA sequencing of the large subunit (LSU) genetic marker with primers LRust1R and LR3 (Vilgalys and Hester 1990; Beenken et al. 2012). The LSU sequence of the rust fungus in South Carolina (GenBank accession: OQ746460) is 99.9% identical to that of Ps. paullula voucher BPI 893085 (763/764 nt.; KY764151), 99.4% identical to that of voucher PIGH 17154 in Florida, USA (760/765 nt.; OQ275201), and 99% identical to that of voucher TNS-F-82075 in Japan (715/722 nt.; OK509071). Based on its morphological and molecular characteristics, the causal agent was identified as Ps. paullula. This pathogen identification was also corroborated by the U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Plant Pathogen Confirmatory Diagnostics Laboratory in Laurel, Maryland. To confirm the fungus's pathogenicity on M. deliciosa and M. adansonii Schott (Sakamoto et al. 2023), three plants of each Monstera species were inoculated by spraying with a suspension of urediniospores collected from the original plant sample (1 × 106 spores per ml; approx. 40 ml per plant). Three non-inoculated control plants of each host species were treated with deionized water in the same manner. Plants were placed in a plastic tray with wet paper towels to maintain moisture. The tray was placed at 22C for an 8-h photoperiod and covered for five days to facilitate infection. On 25 days after inoculation, abundant spots bearing urediniospores were produced on all leaves of inoculated M. deliciosa plants. A few uredinia were observed on two of the three inoculated M. adansonii plants. All non-inoculated control plants remained asymptomatic. Morphological features of urediniospores collected from inoculated plants matched those of Ps. paullula used as the inoculum. Aroid leaf rust on Monstera plants was officially reported in Australia, China, Japan, Malaysia, Philippines, and Florida, USA (Shaw 1991; Sakamoto et al. 2023; Urbina et al. 2023). This is the first report of Ps. paullula causing this disease on M. deliciosa in South Carolina, USA. Monstera species are popular indoor and landscape plants. Potential impact and regulatory responses regarding Ps. paullula, a newly introduced and rapidly spreading pathogen in the USA, warrant further evaluation and discussion.
  19. Qin R, Li Q, Huang S, Chen X, Mo J, Guo T, et al.
    Plant Dis, 2023 Mar 27.
    PMID: 36973906 DOI: 10.1094/PDIS-05-22-1168-PDN
    Persimmon (Diospyros kaki Thunb.) is widely cultivated in China. On October 15, 2019, about 10% of persimmon fruits showed fruit rot in the orchards of Guilin, Guangxi, China (24°45' N, 110°24' E), which could cause more than 15% of yield losses. The initial symptoms of fruit rot exhibited irregular brown to black spots (range from 2 to 4 cm in diameter), the areas surrounding the blackened spots would be soft and rotten, and three diseased fruit samples were collected from three orchards, respectively. Tissues (5×5 mm) were cut from infected margins, surface-disinfected in 75% ethanol for 10 s, 2% NaClO for 2 min, rinsed three times in sterilized distilled water, and incubated on potato dextrose agar (PDA) at 25°C under 12/12 h light/darkness for a week. Forty-one tissues yielded morphologically similar cultures, and three representative isolates LPG1-1, LPG1-2, and YSG-1 were selected from three samples for further study, respectively. Their colonies showed wavy edges, white surfaces, and dense aerial hyphae on PDA after two weeks. Conidia were fusiform, straight to slightly curved, and 4-septate; basal cells were conical, hyaline, thin, and verruculose with two or three long and hyaline apical appendages and one short apical appendage; three median cells of LPG1-1 with length 14.06 to 17.69 μm (n=100), and LPG1-2 with length 14.03 to 17.61 μm (n=100) were dark brown to olivaceous, while three median cells of YSG-1 with length 12.54 to 15.58 μm (n=100) were dark brown. The conidial sizes of LPG1-1, LPG1-2, and YSG-1 were 17.41 to 27.68 × 4.63 to 8.55 μm (n=100), 18.06 to 27.41 × 4.33 to 8.21 μm (n=100), and 16.58 to 27.73 × 4.99 to 8.39 μm (n=100), respectively. The morphological characteristics were consistent with Neopestalotiopsis spp. (Maharachchikumbura et al. 2012; Maharachchikumbura et al. 2014). Primer pairs ITS4/ITS5, BT2a/BT2b, and EF1-526F/EF-1567R were used to amplify internal transcribed spacer (ITS), beta-tubulin (TUB2), and translation elongation factor 1 alpha (TEF1-α), respectively (Shu et al., 2020). All DNA fragments were sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). Sequences have been deposited in GenBank (ITS: OM349120 to OM349122, TUB2: OM688188 to OM688190, TEF1-α: OM688191 to OM688193). Based on BLASTn analysis of ITS, TUB2, and TEF1-α sequences, the LPG1-1 and LPG1-2 showed over 99% similarity to N. saprophytica, and YSG-1 showed over 99% similarity to N. ellipsospora. Phylogenetic analysis of the three isolates was performed with MEGA10 (version 10.0) based on sequences of ITS, TUB2, and TEF1-α using maximum parsimony analysis. The results revealed that LPG1-1 and LPG1-2 were clustered with N. saprophytica, and YSG-1 was clustered with N. ellipsospora. Pathogenicity tests of three isolates were conducted on 72 healthy persimmon fruits with and without wounds, and 9 fruits are for each treatment. The wound was made by a sterilized needle. Fruits were pre-processed with 75% ethanol for 10 s, 1% NaClO for 2 min and rinsed three times in sterile water. Conidial suspensions (10 µL, 106 conidia/mL in 0.1% sterile Tween 20) were inoculated on each site (4 sites/fruit). Control group was treated with 0.1% sterile Tween 20. All inoculated sites were covered with wet cotton. The inoculated fruits were placed in a plastic box to maintain humidity at 28℃. After 5 days, all wounded fruits showed fruit rot, whereas unwounded and control fruits remained asymptomatic, there were significant differences (P<0.05) in aggressiveness between N. saprophytica (average lesion diameter 13.1 mm) and N. ellipsospora (average lesion diameter 14.9 mm). Koch's postulates were fulfilled by re-isolating the causal agents from inoculated fruits. N. ellipsospora was previously reported as an endophyte in D. montana in southern India (Reddy et al. 2016). N. saprophytica could cause leaf spot of Erythropalum scandens and Magnolia sp., and fruit rot of Litsea rotundifolia in China and leaf spot of Elaeis guineensis in Malaysia (Yang et al. 2021, Ismail et al. 2017). To our knowledge, this is the first report of N. ellipsospora and N. saprophytica causing fruit rot on persimmon in the world. The results will provide a foundation for controlling fruit rot caused by pestalotioid fungi on persimmon.
  20. Zakaria L
    Plant Dis, 2023 Mar;107(3):603-615.
    PMID: 35819350 DOI: 10.1094/PDIS-02-22-0358-FE
    Basal stem rot of oil palm caused by Ganoderma boninense is the most serious disease of oil palm in Malaysia, Indonesia, and other oil-palm-producing countries. Economic losses caused by the disease can be up to USD500 million a year. For many years, basal stem rot was found to infect older palm trees of more than 25 to 30 years in age. Only in the 1950s, the disease began to appear in much younger palm trees, 10 to 15 years old, and, in the last decade or so, palm trees as young as 1 year were infected by the disease. The highest incidence occurs in coastal areas of Southeast Asia but the disease has now infected oil palm in inland areas, mainly oil palm planted in peat soils. Disease incidence is also high in areas previously growing coconut or forest. Basal stem rot infection and spread occur through root-to-root contact, and basidiospores that colonize the roots also play a role. In the early stages of infection by G. boninense, the pathogen behaves as a biotroph and later as a necrotroph, secreting cell-wall-degrading enzymes and triggering host defense responses. Genes, gene products, and metabolic pathways involved in oil palm defense mechanisms against G. boninense have been identified and these metabolites have the potential to be used as markers for early detection of the disease. Integrated disease management used to control basal stem rot includes cultural practices, chemical control, and application of biocontrol agents or fertilizers. Early detection tools have also been developed that could assist in management of basal stem rot infections. Development of resistant or tolerant oil palm is still at an early stage; therefore, the existing integrated disease management practices remain the most appropriate methods for managing basal stem rot of oil palm.
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