This study was conducted to identify the causal organism of bark dieback disease of highbush blueberry (Vaccinium corymbosum L.) observed in Korea. Blueberry, a woody plant that is native to North America, belongs to the family Ericaceae and genus Vaccinium. Of the 400 species of blueberry in the world, most are distributed in the tropics of Malaysia and Southeast Asia. Highbush blueberry is abundantly grown in Canada and the United States and has become a popular commercial crop in Korea for products such as jam, wine, and sauce. Bark dieback disease of blueberry was found in Sunchang (<5% incidence), Jeollabuk-do, Korea in July 2009. Typical symptoms of the disease were blight and dieback on the stems with lesions extending along entire branches. Morphological examination revealed that the perithecia were of the globose type with a nipple, 155 to 490 (374.6) μm, and brown on the dead bark. Asci were bitunicate and clavate or cylindrical with dimensions of 63 to 125 × 16 to 20 μm and containing eight ascospores. Ascospores were of the long ovoid type with dimensions of 13.2 to 23.7 (17.98) × 25.4 to 41.1 (33.21) μm. From extracted genomic DNA, the internal transcribed spacer (ITS)-5.8S ribosomal DNA region was amplified with universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). A BLAST search of GenBank with the ITS sequence revealed that the Sunchang isolate (GenBank Accession No. HQ384217) had 99 to 100% sequence identity with the following Botryosphaeria dothidea accessions: FJ517657, AJ938005, FJ478129, FJ171723, and AJ938004. Phylogenetic analysis with the Sunchang isolate, B. dothidea strains, and related species revealed that the B. dothidea isolate and strains comprised a monophyletic group distinguished from other Botryosphaeria spp. including B. ribis, B. parva, B. protearum, B. lutea, B. australis, B. rhodina, B. obtuse, and B. stevensii (2). On the basis of morphological and molecular results, the isolate was identified as B. dothidea (Moug.) Ces. & De Not. A culture of B. dothidea isolate was grown on potato dextrose agar (PDA) for 10 days. A 5-mm plug was inoculated into stem wounds created with a No. 2 cork borer in 20 2-year-old disease-free blueberry plants grown in a greenhouse. Six plants inoculated with only PDA plugs served as noninoculated controls. The wounds were covered with Parafilm. After 3 months, the Parafilm was removed and black lesions were observed at the fungal inoculation sites, while no lesion was observed on the control plants. To complete Koch's postulates, the fungus was reisolated from the lesions and confirmed to be B. Dothidea (1). There is an urgent need to determine the spread of this disease in Korea, estimate the losses, and develop methods for reducing damage through biological and eco-friendly cultural control methods. References: (1) D. Jurc et al. Plant Pathol. 55:299, 2006. (2) B. Slippers et al. Mycologia 96:83, 2004.
Pears (Pyrus pylifolia L.) are cultivated nationwide as one of the most economically important fruit trees in Korea. At the end of October 2019, bleeding canker was observed in a pear orchard located in Naju, Jeonnam Province (34°53'50.54″ N, 126°39'00.32″ E). The canker was observed on trunks and branches of two 25-year-old trees, and the diseased trunks and branches displayed partial die-back or complete death. When the bark was peeled off from the diseased trunks or branches, brown spots or red streaks were found in the trees. Bacterial ooze showed a rusty color and the lesion was sap-filled with a yeasty smell. Trunks displaying bleeding symptoms were collected from two trees. Infected bark tissues (3 × 3 mm) from the samples were immersed in 70% ethanol for 1 minute, rinsed three times in sterilized water, ground to fine powder using a mortar and pestle, and suspended in sterilized water. After streaking each suspension on Luria-Bertani (LB) agar, the plates were incubated at 25°C without light for 2 days. Small yellow-white bacterial colonies with irregular margins were predominantly obtained from all the samples. Three representative isolates (ECM-1, ECM-2 and ECM-3) were subjected to further characterization. These isolates were cultivated at 39 C, and utilized (-)-D-arabinose, (+) melibiose, (+)raffinose, mannitol and myo-inositol but not 5-keto-D-gluconate, -gentiobiose, or casein. These isolates were identified as Dickeya sp. based on the sequence of 16S rRNA (MT820458-820460) gene amplified using primers 27f and 1492r (Heuer et al. 2000). The 16S rRNA sequences matched with D. fangzhongdai strain ND14b (99.93%; CP009460.1) and D. fangzhongdai strain PA1(99.86%; CP020872.1). The recA, fusA, gapA, purA, rplB, and dnaX genes and the intergenic spacer (IGS) regions were also sequenced as described in Van der wolf et al. (2014). The recA (MT820437-820439), fusA (MT820440-820442), gapA (MT820443-820445), purA (MT820446-820448), rplB (MT820449-820451), dnaX (MT820452-820454) and IGS (MT820455-820457) sequences matched with D. fangzhongdai strains JS5, LN1 and QZH3 (KT992693-992695, KT992697-992699, KT992701-992703, KT992705-992707, KT992709-992711, KT992713-992715, and KT992717-992719, respectively). A neighbor-joining phylogenetic analysis based on the concatenated recA, fusA, gapA, purA, rplB, dnaX and IGS sequences placed the representative isolates within a clade comprising D. fangzhongdai. ECM-1 to 3 were grouped into a clade with one strain isolated from waterfall, D. fangzhongdai ND14b from Malaysia. Pathogenicity test was performed using isolate ECM-1. Three two-year-old branches and flower buds on 10-year-old pear tree (cv. Nittaka), grown at the National Institute of Horticultural and Herbal Science Pear Research Institute (Naju, Jeonnam Province in Korea), were inoculated with 10 μl and 2 μl of a bacterial suspension (108 cfu/ml), respectively, after wounding inoculation site with a sterile scalpel (for branch) or injecting with syringe (for flower bud). Control plants were inoculated with water. Inoculated branches and buds in a plastic bag were placed in a 30℃ incubator without light for 2 days (Chen et al. 2020). Both colorless and transparent bacterial ooze and typical bleeding canker were observed on both branches and buds at 3 and 2 weeks post inoculation, respectively. No symptoms were observed on control branches and buds. This pathogenicity assay was conducted three times. We reisolated three colonies from samples displaying the typical symptoms and checked the identity of one by sequencing the dnaX locus. Dickeya fangzhongdai has been reported to cause bleeding canker on pears in China (Tian et al. 2016; Chen et al. 2020). This study will contribute to facilitate identification and control strategies of this disease in Korea. This is the first report of D. fangzhongdai causing bleeding canker on pears in Korea.
Jackfruit (Artocarpus heterophyllus) is an important tropical commercial fruit crop grown in Hainan province, China. In recent years, severe jackfruit bronzing disease has been found in 11 cities and counties in Hainan. On average, 80% of trees in a jackfruit orchard are affected once bronzing disease is detected. The disease is characterized by yellow-orange to reddish discoloration of the pulp and rags of infected fruit (Hernández-Morales et al. 2017). Jackfruit bronzing disease has been reported previously in the Philippines (Gapasin et al. 2012), Malaysia (Zulperi et al. 2017), and Mexico (Hernández-Morales et al. 2017). Diseased samples of jackfruit 'Tai Eight' with the bronzing symptoms were collected from a plantation in Changjiang, Hainan. The samples were sterilized with 75% ethanol for 30 s, then soaked with 1% sodium hypochlorite for 8 min, and rinsed with sterilized distilled water. The sterilized tissues were ground in 2 mL sterile water, and allowed to stand for 30 min. Then, 500 μL of the supernatant was spread on Glucose-Yeast agar medium and incubated overnight at 28ºC. Representative bacterial colonies were lemon-yellow, convex and smooth, transparent with entire edges. Colonies were Gram-negative, positive for catalase and gelatin liquefaction, which were consistent with the characteristics of P. stewartii subsp. stewartii. In PCR amplifications, an 920 bp amplicon of strain JTPE2 with the primers ES16/ESIG2c (Coplin et al. 2002) and an 1100 bp amplicon of strain JTPC2 with the primers CPSL1/CPSR2c (Ibrahim et al. 2019) were obtained, whereas no bands were observed for the negative control samples. The ES16/ESIG2c and CPSL1/CPSR2c fragments were sequenced for nucleotide BLAST (BLASTn) searches of the NCBI database and phylogenetic tree construction. The obtained ES16/ESIG2c sequences (SAR accession no. SRR22405292) showed 99.07%-99.60% similarity with P. stewartii subsp. stewartii (CP017581, AJ311838 and MF598163). The obtained CPSL1/CPSR2c sequences (SAR accession no. SRR22405293) showed 99.40%-99.99% similarity with P. stewartii subsp. stewartii (MW971422, MH752485 and MH257287). Phylogenetic analysis based on cpsDE sequences (Ibrahim et al. 2019) using the maximum likelihood method revealed that strains JTPE2 and JTPC2 were clustered together with P. stewartii subsp. stewartii. A pathogenicity test was conducted by injecting 2 mL of 108 CFU/ml bacterial suspension into pulp from healthy, surface-sterilized jackfruit. Pulp injected with sterilized distilled water served as a negative control. All inoculated samples produced bronzing symptoms from 2-3 weeks post-inoculation similar to the field-observed symptoms, whereas control fruit were asymptomatic. The strains were reisolated from symptomatic jackfruit pulp to complete Koch's postulates. The bacterial suspension was inoculated on 2-week-old maize seedlings to supplement in vivo pathogenicity testing. Typical Stewart's disease leaf symptoms were visible at 2 weeks post-inoculation. Based on morphological, biochemical, and physiological evidence, pathogenicity tests, and molecular analyses, the pathogenic bacterium isolated from 'Tai Eight' jackfruit was identified as P. stewartii subsp. stewartii. To our knowledge, this is the first report of bronzing disease caused by P. stewartii subsp. stewartii on jackfruit in China, which may assist in preventing the global spread of jackfruit bronzing disease.
Multiple diseases, including brown spot (Cochliobolus miyabeanus), leaf spot (Epicoccum sorghimum), and blast (Magnaporthe oryzae), can cause spot-like symptoms on the leaves of rice. In July 2021, a disease showing symptoms like brown spot was observed in an 8-hectare field of rice, with disease incidence of >30%, in Beaumont, Texas. Lesions started as small pinhead-size blackish spots on leaf tips or from the edges of leaf blades. The spots enlarged to become irregular (most) or oval brown spots with a slight chlorotic halo. Diseased leaves were collected, washed in running tap water and cut into small pieces. Pieces of the tissue were surface sterilized with 1%NaOCl for 2 min followed by 75% ethanol for 30 s and then washed in sterile distilled water three times with each time lasting for 1 min. The disinfected tissue pieces were air dried, placed on potato dextrose agar (PDA) medium and incubated at 25℃. Initially fungal colonies were hairy in texture with light dark brown center and whitish edge and dark brown pigmentation at the reverse side. Mature colonies turned to black in the center and dark brown toward the edge, with black at the reverse side after 2 or more weeks of incubation. Conidia were oval to narrowly oblong, rounded at the ends, with 2 to 6 distoseptate, and 15 to 35 × 6 to 10 µm in size. These morphological characteristics were similar to those described for Curvularia hawaiiensis (Aslam et al. 2019; Ellis 1971; Kusai et al. 2015). For molecular identification, DNA was extracted and the two different rRNA regions internal transcribed spacer (ITS) and large subunit (LSU), and the two genes RNA Polymerase II (RPB1) and translation elongation factor 1 alpha (EF1) of the fungus were amplified using the primers of ITS1/ITS4 (Wang et al. 2014), D1/D2 domain region of LSU (Fell et al. 200), and RPB1 and EF1 (Wang et al. 2014), respectively, and sequenced. The ITS sequence (OK397200) was 98.27% identical to C. hawaiiensis (KP131943); the EF1 sequence (OK492159) was 99.78% identical to C. hawaiiensis (KC503942); the LSU sequence (OK397295) was 98.96% identical to multiple C. hawaiiensis (MN055715, MH160813, MH875853, etc.); the RPB1 sequence (OK492160) was 97.41% identical to C. hawaiiensis (JN992363). To evaluate pathogenicity, three rice plants (cv. Presidio) at the 3-leaf stage were spray inoculated with a conidial suspension of 1 x 106 conidia/ml. Another set of three plants that were sprayed with sterilized distilled water served as the controls. Treated plants were maintained in a greenhouse with temperature ranging from 25 to 30℃. After 2 weeks, typical symptoms, like those observed in the field, developed on the inoculated plants while no symptoms developed on the control plants. The same fungus was consistently re-isolated from the diseased plants. The pathogenicity test was conducted three times with similar results. To our knowledge, this is the first report of brown leaf spot caused by C. hawaiiensis in rice in the United States. Curvularia species are frequently associated with rice grain and cause blackish discoloration symptoms on grain kernels. Recently, however, C. hawaiiensis has also been reported to cause brown leaf spot in Malaysia (Kusai et al. 2015) and Pakistan (Aslam et al. 2019). This research will help identify this disease from other leaf spot-like diseases and develop effective management strategies.
Cabbage (Brassica oleracea L. var. capitata L.) is one of the most important vegetables cultivated in Pahang and Kelantan, Malaysia. Pectobacterium carotovorum can cause soft rot on a wide range of crops worldwide, especially in countries with warm and humid climates such as Malaysia. Cabbage with symptoms of soft rot from commercial fields were sampled and brought to the laboratory during the winter of 2010. Disease symptoms were a gray to pale brown discoloration and expanding water-soaked lesions on leaves. Several cabbage fields producing white cultivars were investigated and 27 samples were collected. Small pieces of leaf samples were immersed in 5 ml of saline solution (0.80% NaCl) for 20 min to disperse the bacterial cells. Fifty microliters of the resulting suspension was spread on nutrient agar (NA) and King's B medium and incubated at 30°C for 48 h. Purification of cultures was repeated twice on these media. Biochemical and phenotypical tests gave these results: gram negative, rod shaped, ability to grow under liquid paraffin (facultative anaerobe); oxidase negative; phosphatase negative; positive degradation of pectate; sensitive to erythromycin; negative to Keto-methyl glucoside utilization, indole production and reduction sugars from sucrose were negative; acid production from sorbitol and arabitol was negative and from melibiose, citrate, and raffinose was positive. Hypersensitivity reaction on tobacco leaf with the injection of 106 CFU/ml of bacterial suspension for all strains was positive. Four representative strains were able to cause soft rot using cabbage slices (three replications) inoculated with a bacterial suspension at 106 CFU/ml. Inoculated cabbage slices were incubated in a moist chamber at 80% relative humidity and disease symptoms occurred after 24 h. Cabbage slices inoculated with water as a control remained healthy. The bacteria reisolated from rotted cabbage slices on NA had P. carotovorum cultural characteristics and could cause soft rot in subsequent tests. PCR amplification with Y1 and Y2 primers (1), which are specific for P. carotovorum, produced a 434-bp band with 15 strains. PCR amplification of the 16S-23S rRNA intergenic transcribed spacer region (ITS) using G1 and L1 primers gave two main bands approximately 535 and 580 bp and one faint band approximately 740 bp when electrophoresed through a 1.5% agarose gel. The ITS-PCR products were digested with RsaI restriction enzyme. According to biochemical and physiological characterictics (2), PCR-based pel gene (1), and analysis by ITS-PCR and ITS-restriction fragment length polymorphism (3), all isolates were identified as P. carotovorum subsp. carotovorum. This pathogen has been reported from Thailand, Indonesia, and Singapore with whom Malaysia shares its boundaries. To our knowledge, this is the first report of P. carotovorum subsp. carotovorum in cabbage from Malaysia. References: (1) A. Darraas et al. Appl. Environ. Microbiol. 60:1437, 1994. (2) N. W. Schaad et al. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, 2001. (3) I. K. Toth et al. Appl. Environ. Microbiol. 67:4070, 2001.
Elaeagnus conferta Roxb. is a perennial evergreen climbing shrub and is mainly native to India, Vietnam, Malaysia, and South China (Gupta & Singh, 2021). Various parts of this plant are used to treat multiple diseases(Gupta et al., 2021). Between during the months of March and April of 2021, in Kunming city of grower fields, Yunnan Province (N 25°02'; E 102°42'), southwest China. Some postharvest E. conferta fruits showed brown spots of decay with a greyish mycelium, which symptom only appears on fruit, and did not find it on this plant. The incidence of this disease in postharvest E. conferta fruits ranges from 45 % to 65 % in natural conditions. This pathogen is harmful and causes many plant diseases. Such as rice, oriental persimmon, pear, panicles of mango, and so on (Cho & Shin, 2004; Guillén-Sánchez et al., 2007; Lee et al., 2009). The infected fruit samples surface was disinfected with 75 % ethanol and 0.3 % NaClO for 30 s and 2 min respectively, then aseptic water washing three times. The fruit tissue is rich in carbohydrates and water content, which aid the growth of fungal species. Putting these diseased tissues on a potato dextrose agar (PDA) medium, cultured at 25 ± 1 ℃ for 7 days. The colonies grow on the PDA medium, then separated and puried again. Three pure cultures (YNGH01, YNGH03, YNGH05) were obtained, which were stored in 15 % glycerol at -80 ℃ refrigerator in the State Key Laboratory for Conservation and Utilization of Bio-Resources, Yunnan Agricultural University. After 7 days of cultivation, the colonies were round and the diameter attained up to 38 mm, the surface of the colony showed tiled, fluffy, with a velvet-like texture, greyish-green to smoke-gray, slightly raised in the middle, the edges were radial hollow and wrinkle (Fig. 1A). Conidiophores were solitary, erect, unbranched or rarely branched, slightly flexuous at the apex, septate, dark brown, 254 to 680 µm long, 3.6 to 4.5 µm wide, top of the conidiophores or the rostral were slightly swollen (Fig. 1B). Conidia were light gray or grey, solitary or bispora, irregular in shape and size (Fig. 1C), nearly circular (3.21 × 3.31 µm), oval to lemon-shaped (6.59 × 3.21 µm) or elliptical (8.35 × 4.65 µm). The CTAB method extracts 3 isolates (YNGH01, YNGH03, YNGH05) genomic DNA (Aboul-Maaty & Oraby, 2019). To confirm identity with molecular identification, performed by three different genomic DNA regions, fragments of internal transcribed spacer (ITS), partial translation elongation factor-1 alpha (TEF-1α), and actin (ACT) genomic regions. These genomic DNA were amplified with primers ITS1/4, EF1-728F/986R, and ACT-512F/783R, respectively (Carbone & Kohn, 1999). The sequences of these isolates were uploaded to GenBank (YNGH01: ON753810, ON868696, ON912090 YNGH03: ON753812, ON868698, ON912092, and YNGH05: ON753814, ON868700, ON912094). NCBI's BLASTn search of those ITS sequences showed 99.81% similar to C. tenuissimum (MG873077.1), and sequences TEF-1α and ACT were 100% identical to several isolates of C. tenuissimum (OM256526.1 and MT154171.1). Combined the ITS region, TEF-1α, and actin (ACT) genomic regions of isolates YNGH01, YNGH03 and YNGH05 to construct a phylogenetic tree with MEGA11. Maximum likelihood phylogenetic analyses further confirmed the results (Fig. 2)(Santos et al., 2020). Healthy and mature E. conferta fruits were used for pathogenicity test. Pathogens were washed with sterilized water at a final concentration of 2× 106 spores/mL (Jo et al., 2018). The test was divided into A and B groups (A: The surface of fruits was pierced with a sterilized needle that carried pathogenic fungus of final concentration at 2×106 spores/mL B: Sprayed at the concentration of 2×106 spores/mL on fruits). The control fruits were treated with sterilized water and stored at 25 ± 1 ℃ with a relative humidity of 80 %, average group with 10 fruits in this test, which was repeated three times. After 7 days, the fruits of group A were initially sesame seed size of the disease spots, nearly round, irregular, with grayish-brown spots, and slightly depressed. Later, the lesion gradually turns dark brown (Fig. 1D). And group B began with small patches of brown fungal growth on the pericarp, with the development of the disease, the necrotic spots enlarged and developed irregular and coalesced, the color of spots became gray or black gradually (Fig. 1E). The symptoms were similar to previously observed and the pathogen was reisolated and identified as C. tenuissimum. Control fruits were healthy (Fig. 1F). The pathogens test fulfilled Koch's postulates. According to morphology (Bensch et al., 2012), rDNA-ITS, TEF-1α, and ACT sequence analysis, phylogenetic analysis, and pathogenicity test, the pathogen was identified as C. tenuissimum. To our knowledge, this is the first report of C. tenuissimum occurring on E. conferta fruits in China.
Banana is the second largest cultivated fruit crop in Malaysia, and is cultivated for both the domestic market and also for export. Anthranose is a well-known postharvest disease of banana and with high potential for damaging market value, as infection commonly occurs during storage. Anthracnose symptoms were observed on several varieties of banana such as mas, berangan, awak, nangka, and rastali in the states of Perak and Penang between August and October 2011. Approximately 80% of the fruits became infected with initial symptoms characterized as brown to black spots that later became sunken lesions with orange or salmon-colored conidial masses. Infected tissues (5 × 5 mm) were surface sterilized by dipping in 1% sodium hypochlorite (NaOCl) for 3 to 5 min, rinsed with sterile distilled water, and plated onto potato dextrose agar (PDA). Direct isolation was done by transferring the conidia from conidial masses using an inoculation loop and plating onto PDA. For both methods, the PDA plates were incubated at 27 ± 1°C with cycles of 12 h light and 12 h darkness. Visible growth of mycelium was observed after 4 to 5 days of incubation. Twenty isolates with conidial masses were recovered after 7 days of incubation. The isolates produced grayish white to grayish green and grey to moss dark green colony on PDA, pale orange conidial masses, and fusiform to cylindrical and hyaline conidia with an average size of 15 to 19 × 5 to 6 μm. Appresoria were ovate to obovate, dark brown, and 9 to 15 × 7 to 12 μm and setae were present, slightly swollen at the base, with a tapered apex, and brown. The cultural and morphological characteristics of the isolates were similar to those described for C. gleosporioides (1,2,3). All the C. gloeosporioides isolates were deposited in culture collection at Plant Pathology Lab, University Sains Malaysia. For confirmation of the identity of the isolates, ITS regions were sequenced using ITS4 and ITS5 primers. The isolates were deposited in GenBank with accessions JX163228, JX163231, JX163201, JX163230, JX163215, JX163223, JX163219, JX163202, JX163225, JX163222, JX163206, JX163218, JX163208, JX163209, JX163210, JX431560, JX163212, JX163213, JX431540, and JX431562. The resulting sequences showed 99% to 100% similarity with multiple C. gloeosporioides isolates in GenBank. Pathogenicity tests were conducted using mas, berangan, awak, nangka, and rastali bananas. Fruit surfaces were sterilized with 70% ethanol and wounded using a sterile scalpel. Two inoculation techniques were performed separately: mycelia plug and conidial suspension. Mycelial disc (5 mm) and a drop of 20 μl spore suspension (106 conidia/ml) were prepared from 7-day-old culture and placed on the fruit surface. The inoculated fruits were incubated at 27 ± 1°C for 10 days at 96.1% humidity. After 3 to 4 days of inoculation, brown to black spotted lesions were observed and coalesced to become black sunken lesions. Similar anthracnose symptoms were observed on all banana varieties tested. C. gloeosporioides was reisolated from the anthracnose lesions of all the inoculated fruit in which the cultural and morphological characteristics were the same as the original isolates. To our knowledge, this is the first report of C. gloeosporioides causing anthracnose of Musa spp. in Malaysia. References: (1) P. F. Cannon et al. Mycotaxon 104:189, 2008. (2) J. E. M. Mordue. Glomerella cingulata. CMI Description of Pathogenic Fungi and Bacteria, No. 315. CAB International,1971. (3) H. Prihastuti et al. Fungal Diversity 39:89, 2009.
Anthracnose fruit rot caused by various Colletotrichum spp. is a serious disease for pepper (Capsicum annuum) growers, resulting in extensive fruit loss (Harp et al. 2008). Samples of five pepper fruits were obtained from two commercial farms in Lexington and Pickens counties, South Carolina, in August and September 2019, respectively. All fruits had two or more soft, sunken lesions covered with salmon-colored spore masses. Pieces of diseased tissue cut from the margins of lesions were surface disinfested in 0.6% sodium hypochlorite, rinsed in sterile deionized water, blotted dry, and placed on one-quarter-strength potato dextrose agar (PDA/4) amended with 100 mg chloramphenicol, 100 mg streptomycin sulfate, and 60.5 mg mefenoxam (0.25 ml Ridomil Gold EC) per liter. Two isolates of Colletotrichum sp. per fruit were preserved on dried filter paper and stored at 10º C. One additional isolate of Colletotrichum sp. had been collected from a jalapeño pepper fruit on a farm in Charleston County, South Carolina, in 1997. Colony morphology of three isolates, one per county, on Spezieller Nährstoffarmer Agar (SNA) was pale grey with a faint orange tint. All isolates readily produced conidia on SNA with an average length of 16.4 μm (std. dev. = 1.8 μm) and a width of 2.2 μm (std. dev. = 0.2 μm). Conidia were hyaline, smooth, straight, aseptate, cylindrical to fusiform with one or both ends slightly acute or round, matching the description of C. scovillei (Damm et al. 2012). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-tubulin (TUB2) genes from three isolates were amplified and sequenced with the primer pairs GDF1/GDR1 and T1/Bt2b, respectively. Species within the C. acutatum clade can be readily distinguished with GAPDH or TUB2 (Cannon et al. 2012). The GAPDH and TUB2 sequences for all three isolates were 100% similar to each other and strain CBS 126529 (GAPDH accession number JQ948597; TUB2 accession number JQ949918) of C. scovillei (Damm et al. 2012). GAPDH and TUB2 sequences for each isolate were deposited in GenBank under the accessions MT826948-MT826950 and MT826951-MT826953, respectively. A pathogenicity test was conducted on jalapeño pepper fruits by placing a 10-ul droplet of a 5 x 105 conidial suspension of each isolate onto a wound made with a sterile toothpick. Control peppers were mock inoculated with 10 ul sterile distilled water. A humid chamber was prepared by placing moist paper towels on the bottom of a sealed crisper box. Inoculated peppers were placed on upside-down 60 ml plastic condiment cups. Three replicate boxes each containing all four treatments were prepared. The experiment was repeated once. After 7 days in the humid chamber at 26ºC, disease did not develop on control fruits, whereas soft, sunken lesions covered with salmon-colored spores developed on inoculated fruits. Lesions were measured and C. scovillei was re-isolated onto amended PDA/4 as previously described. Lesion length averaged 15.6 mm (std dev. = 4.1 mm) by 11.5 mm (std dev. = 2.0 mm). Colletotrichum sp. resembling the original isolate were recovered from all inoculated fruit, but not from non-inoculated fruit. C. scovillei has been reported in Brazil in South America and in China, Indonesia, Japan, Malaysia, South Korea, Taiwan, and Thailand in Asia (Farr and Rossman 2020). This is the first report of C. scovillei as the casual organism of anthracnose fruit rot on pepper in South Carolina and the United States.
Production of watermelon (Citrullus lanatus) in Malaysia was 150,000 mt in 2020 (Malaysian Department of Agriculture, 2021). In November 2019, nine locally produced watermelon fruit (red flesh, seedless) from five local stores in the states of Kelantan, Terengganu, and Penang exhibited sunken, circular, brown lesions that enlarged to1.5 to 10 cm in diameter with scattered orange masses of conidia. Lesions coalesced to cover approximately 50% of the fruit surface. Lesions were surface sterilized by spraying 70% alcohol onto the fruit followed by drying with sterilized paper towels. A total of 153 tissue segments (1×1 cm) were excised from the rind, immersed in 1% sodium hypochlorite for 3 min, rinsed twice for 1 min in sterilized distilled water, air-dried, transferred to potato dextrose agar (PDA) plates, and incubated at 25±1°C for 7 days. Single-spore transfers produced pure cultures, resulting in 12 isolates. Colonies on PDA were initially white and turned pale gray with age. Conidia were hyaline, one end round and the other narrowly acute, aseptate, smooth-walled, straight, cylindrical to clavate, 10.5-16.5 µm × 3-4.5 μm (n = 30). Observed morphological characters matched published description of Colletotrichum spp. (Damm et al. 2012). Internal transcribed spacer (ITS) and glyceraldehyde-phosphate dehydrogenase (GAPDH) genes were amplified using primer sets ITS1/ITS4 and GDF1/GDF2, respectively. All sequences were deposited in GenBank (MW856808 for ITS; MZ219296 for GAPDH). A BLASTn search of both sequences on GenBank showed 99% identity with C. scovillei along with other closely related Colletotrichum species. Phylogenetic analysis of ITS and GAPDH alignments, using maximum likelihood along with reference strains of closely related species from Mycobank, confirmed species identity as C. scovillei. A pathogenicity test was conducted on two healthy watermelon fruit (red flesh, seedless). A 6-mm-diameter mycelial plug of a colony on PDA was positioned on a 0.5-cm-long wound on each fruit; a sterile PDA plug placed on a similar wound on the opposite side served as a control. Fruit were incubated at 25±1°C for 7 days in plastic-wrapped trays above distilled water to maintain high humidity. Small, sunken, circular brown lesions appeared and expanded at inoculation sites within 7 days. Symptoms were identical to those produced by natural infections, and the controls were asymptomatic. Isolates from the lesions at the inoculation sites were confirmed as C. scovillei based on morphological characteristics, fulfilling Koch's postulates. The pathogenicity test was conducted four times with a total of eight fruit. Many species in the C. orbiculare complex cause watermelon anthracnose (Keinath, 2018). To our knowledge, this is the first report of C. scovillei (C. acutatum species complex; Damm et al. 2012) causing anthracnose on watermelon in Malaysia. Anthracnose caused by C. scovillei has been confirmed on other crops such as pepper (Toporek and Keinath, 2021), banana (Zhou et al., 2017), and chili (Oo et al., 2017). This insight will inform efforts to improve management of watermelon anthracnose in Malaysia.
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.
Plumeria alba L. is a flowering plant in the family Apocynaceae and widely cultivated in Malaysia as a cosmopolitan ornamental plant. In January 2020, anthracnose lesions were observed on leaves of Plumeria alba planted in Agricultural Farm, Universiti Putra Malaysia, in Selangor state, Malaysia. The disease mainly affected the leaves with symptoms occurring with approximately a 60% disease incidence. Ten symptomatic leaves were sampled from 3 different trees in the farm. Symptoms initiated as small circular necrotic spots that rapidly enlarged into black lesions with pale brown borders. Diseased tissues (5×5 mm) were surface-sterilized with 70% ethanol for 1 min, rinsed three times with sterile distilled water, dried on sterile filter papers, plated on PDA and, incubated at 25 °C with a 12-h photoperiod. A total of seven single-spore isolates with similar colony morphologies were obtained from tissue samples. After 7 days, the colonies raised the entire margin and showed white-to-gray aerial mycelium, orange conidial masses in the center and appeared dark brown at the center of the reverse view. The conidia were 1-celled, hyaline, smooth-walled, cylindrical with narrowing at the center, averaged (13-15 μm × 3 - 4 μm) (n=40) in size. Morphological characteristics of the isolates were similar to those detailed in taxonomic description of Colletotrichum sp. (Prihastuti et al. 2009). For molecular identification, genomic DNA of two representative isolates, PL3 and PL4 was extracted from fresh mycelium using DNeasy Plant Mini Kit (Qiagen, USA). The internal transcribed spacer (ITS) region, actin (ACT) and calmodulin (CAL) genes were amplified using ITS5/ITS4 (White et al. 1990), ACT-512F/783R (Carbone and Kohn 1999) and CL1C/CL2C primer sets (Weir et al. 2012). A BLAST nucleotide search of GenBank using ITS sequences showed 100% identity to Colletotrichum siamense ex-type culture ICMP 18578 (GenBank accession no. JX010171). ACT and CAL sequences showed 100% identity with C. siamense ex-type isolate BPD-I2 (GenBank accession no. FJ907423 and FJ917505). The sequences were deposited in GenBank (ITS: accession nos. MW335128, MT912574), ACT: accession nos. MW341257, MW341256, CAL: accession nos. MW341255 and MT919260). Based on these morphological and molecular characteristics, the fungus was identified as C. siamense. Pathogenicity of PL3 and PL4 isolates was verified using four healthy detached leaves of Plumeria alba. The leaves were surface-sterilized using 70% ethanol and rinsed twice with sterile water before inoculation. The leaves (three inoculation sites/leaf) were wounded by puncturing with a sterile needle through the leaf cuticle and inoculated in the wound site with 10-μl of conidial suspension (1×106 conidia/ml) from 7-days-old culture on PDA. Four leaves were used as a control and were inoculated only with 10-μl of sterile distilled water. Inoculated leaves were kept in humid chambers for 2 weeks at 25 °C with 98% relative humidity on a 12-h fluorescent light/dark period. The experiment was repeated three times. Anthracnose symptoms were observed on all inoculated leaves after 3 days, whereas controls showed no symptoms. Fungal isolates from the diseased leaves showed the same morphological characteristics as isolates PL3 and PL4, confirming Koch's postulates. C. siamense has been reported causing anthracnose on rose (Rosa chinensis) in China (Feng et al. 2019), Coffea arabica in Thailand (Prihastuti et al. 2009) and mango leaf anthracnose in Vietnam (Li et al. 2020). To our knowledge, this is the first report of Colletrotrichum siamense causing leaf anthracnose on Plumeria alba in Malaysia. Accurate identification of this pathogen provides a foundation in controlling anthracnose disease on Plumeria alba.
Rice (Oryza sativa L.) has been farmed in Malaysia since ancient times and is one of the most important commercial crops (Ma'arup et al. 2020). Throughout January to August 2022, chlorotic spots with brown halos ranging 2 to 10 mm wide were found on upper leaves of rice variety Mahsuri in the vegetative stage with a severity and incidence of approximately 60% and 100%, respectively in Kampung Tagas, Sabah, Malaysian Borneo (06°09'41.8"N, 116°13'45.1"E). As the disease developed, the spots coalesced into larger chlorotic spots. Three leaf pieces (5 x 5 mm) were excised from lesion margins, surface sterilized based on Khoo et al. (2022a), before plating on water agar (WA) at 25°C. Purification of fungi was conducted on WA using hyphal tip isolation. When three pure cultures were obtained, the fungi were cultured on potato dextrose agar (PDA) and WA for 7 days in 12 h light and 12 h dark at 25°C for the macro- and micro-morphological characterization, respectively. The colonies of the three isolates on PDA were initially gray, later becoming dark. Conidia (n=30) were fusiform, smooth-walled, dark-brown, and melanized with three transverse septa, measuring 7.3 to 11.4 × 16.2 to 27.2 µm. The isolates were named Tagas01, Tagas02, Tagas03. Genomic DNA was extracted from fresh mycelia of the pathogens based on the extraction method described by Khoo et al. (2022b). The primers ITS1/ITS4 (White et al. 1990), GPD1/GPD2 (Berbee et al. 1991), and EF1-983F/EF1-2218R (Schochet al. 2009) were used to amplify the internal transcribed spacer (ITS) region of rDNA, partial fragments of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor (EF-1α) region, respectively based on PCR conditions as described previously (Khoo et al. 2022a). The sequences were deposited in GenBank under accession numbers OP268402, OP271304, OP271305 (677/677 bp) (ITS), OP270699, OP270703, OP270704 (609/613 bp) (GAPDH), OP270700-OP270702 (928/930 bp) (EF-1α). They were 99.35-100% similar to the Curvularia lunata ITS (HF934911), GAPDH (LT715821), and Curvularia dactyloctenicola EF-1α (MF490858) type sequences. Although C. dactyloctenicolais related to C. lunata, the conidia of the former are much wider making them easier to differentiate (Marin-Felix et al. 2017). Phylogenetic analysis using maximum likelihood based on the combined ITS, GAPDH and EF-1α sequences indicated that the isolate formed a supported clade to C. lunata. The pathogens were identified as C. lunata based on morphological and molecular characterization. Koch's postulates were performed. Three replicate healthy rice at the vegetative stage were sprayed with a spore suspension of 1 × 106 spore/ml in distilled sterilized water, prepared from 1-week-old fungal culture, grown in the dark on WA. Three replicate rice plants were sprayed with distilled sterilized water as control. Plants were covered with transparent polyethylene bags to keep moisture, and kept in a greenhouse at ~27°C. Bags were removed after 4 days of incubation. Monitoring and incubation were performed in greenhouse based on Iftikhar et al. (2022). The pathogenicity test was also performed using isolate Tagas02 and Tagas03. All inoculated leaves developed symptoms as described after 6 days post-inoculation, whereas no symptoms occurred on controls. The experiments were repeated twice. The reisolated fungi were identical to the pathogen morphologically and molecularly, thus fulfilling Koch's postulates. C. lunata has been reported in Peninsular Malaysia (Lee et al. 2012). This is the first report of C. lunata causing leaf spot on Oryza sativa in Sabah, Malaysian Borneo. This illness not only reduces yields and lowers milling quality, but it may also be mistaken for rice blast, necessitating needless fungicide spraying.
Red-fleshed dragon fruit (Hylocereus polyrhizus [Weber] Britton & Rose) is a newly introduced and potential crop in the Malaysian fruit industry. Besides its nutritious value, the fruit is being promoted as a health crop throughout Southeast Asia. In April of 2007, a new disease was observed in major plantations of H. polyrhizus throughout five states (Kelantan, Melaka, Negeri Sembilan, Penang, and Perak) in Malaysia with 41 and 25% disease incidence and severity, respectively. Stems of H. polyrhizus showed spots or small, circular, faint pink-to-beige necrotic lesions that generally coalesced as symptoms progressed. Symptom margins of diseased stem samples were surface sterilized with a 70% alcohol swab, cut into small blocks (1.5 × 1.5 × 1.5 cm), soaked in 1% sodium hypochlorite (NaOCI) for 3 min, and rinsed in several changes of sterile distilled water (each 1 min). The surface-sterilized tissues were placed onto potato dextrose agar (PDA) and incubated under alternating 12-h daylight and black light for 7 days. A fungus was consistently isolated from the stems of symptomatic H. polyrhizus and identified as Curvularia lunata (Wakker) Beodijn (1-3) that showed pale brown multicelled conidia (phragmoconidia; three to five celled) that formed apically through a pore (poroconidia) in sympodially, elongating, geniculated conidiophores. Conidia are relatively fusiform, cylindrical, or slightly curved, with one of the central cells being larger and darker (26.15 ± 0.05 μm). All 25 isolates of C. lunata obtained from diseased H. polyrhizus are deposited at the Culture Collection Unit, Universiti Sains Malaysia and available on request. Isolates were tested for pathogenicity by injecting conidial suspensions (1 × 106 conidia/ml) and pricking colonized toothpicks on 25 healthy H. polyrhizus stems. Controls were treated with sterile distilled water and noncolonized toothpicks. All inoculated plants and controls were placed in a greenhouse with day and night temperatures of 30 to 35°C and 23 to 30°C, respectively. Development of external symptoms on inoculated plants was observed continuously every 2 days for 2 weeks. Two weeks after inoculation, all plants inoculated with all isolates of C. lunata developed stem lesions similar to those observed in the field. No symptoms were observed on the control plants and all remained healthy. C. lunata was reisolated from 88% of the inoculated stems, completing Koch's postulates. The pathogenicity test was repeated with the same results. To our knowledge, this is the first report of C. lunata causing a disease on H. polyrhizus. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, England, 1971. (2) R. R. Nelson and F. A. Hassis. Mycologia 56:316, 1964. (3) C. V. Subramanian. Fungi Imperfecti from Madras V. Curvularia. Proc. Indian Acad. Sci. 38:27, 1955.
Indian spinach (Basella rubra L.) is a red stem species of Basella that is cultivated worldwide as an ornamental and the aerial parts are also consumed as a vegetable. In May of 2011, symptoms of damping-off were observed on approximately 10% of the plants at the stem base around the soil line of seedlings in a greenhouse in Homestead, FL. Lesions were initially water soaked, grayish to dark brown, irregular in shape, and sunken in appearance on large plants, causing the infected seedlings to collapse and eventually die. Symptomatic stem tissue was surface sterilized with 0.6% sodium hypochlorite, rinsed in sterile distilled water, air dried, and plated on potato dextrose agar (PDA). Plates were incubated at 25°C in darkness for 3 to 5 days. A fungus was isolated in all six isolations from symptomatic tissues on PDA. Fungal colonies on PDA were light gray to brown with abundant growth of mycelia, and the hyphae tended to branch at right angles when examined under a microscope. A septum was always present in the branch of hyphae near the originating point and a slight constriction at the branch was observed. Neither conidia nor conidiophores were found from the cultures on PDA. The characteristics of hyphae, especially the right angle branching of mycelia, indicate close similarity to those of Rhizoctonia solani (2,3). The internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced (GenBank Accession No. JN545836). Subsequent database searches by the BLASTN program indicated that the resulting sequence had a 100% identity over 472 bp with the corresponding gene sequence of R. solani anastomosis group (AG) 4 (GenBank Accession No. JF701752.1), a fungal pathogen reported to cause damping-off on many crops. Pathogenicity was confirmed through inoculation of healthy India spinach plants with the hyphae of isolates. Four 4-week-old plants were inoculated with the isolates by placing a 5-mm PDA plug of mycelia at the stem base and covering with a thin layer of the soil. Another four plants treated with sterile PDA served as a control. After inoculation, the plants were covered with plastic bags for 24 h and maintained in a greenhouse with ambient conditions. Four days after inoculation, water-soaked, brown lesions, identical to the symptoms described above, were observed on the stem base of all inoculated plants, whereas no symptoms developed on the control plants. The fungus was isolated from affected stem samples, and the identity was confirmed by microscopic appearance of the hyphae and sequencing the ITS1/ITS4 intergenic spacer region, fulfilling Koch's postulates. This pathogenicity test was conducted twice. R. solani has been reported to cause damping-off of B. rubra in Ghana (1) and Malaysia (4). To our knowledge, this is the first report of damping-off caused by R. solani AG-4 on Indian spinach in Florida and the United States. With the increased interest in producing Asian vegetables for food and ornamental purposes, the occurrence of damping-off on Indian spinach needs to be taken into account when designing programs for disease management in Florida. References: (1) H. A. Dade. XXIX. Bull. Misc. Inform. 6:205, 1940. (2) J. R. Parmeter et al. Phytopathology 57:218, 1967. (3) B. Sneh et al. Identification of Rhizoctonia species. The American Phytopathological Society, St Paul, MN, 1991. (4) T. H. Williams and P. S. W. Liu. Phytopathol. Pap. 19:1, 1976.
In January 2011, branch samples were collected from langsat (Lansium domesticum Corr.), a fruit from Southeast Asia with an expanding niche market in Hawaii, exhibiting corky bark symptoms similar to that found on rambutan (Nephelium lappaceum) and litchi (Litchi chinensis) (3). The orchard, located along the Hamakua Coast of Hawaii Island, had 5- to 10-year-old trees, all with corky bark symptoms. As the trees matured, the cankers increased in size and covered the branches and racemes, often resulting in little to no fruit production. Scattered along the infected bark tissue were elongated, black ascomata present in the cracks. Ascomata were removed from the cracks using a scalpel blade, placed at the edge of a water agar petri dish and gently rolled along the agar surface to remove bark tissue and other debris. Individual ascomata were placed in 10-μl drops of 10% sodium hypochlorite on fresh water agar for 20 s, removed, and placed on potato dextrose agar petri dishes amended with 25 μg/ml streptomycin. The isolates were kept at 24°C under continuous fluorescent lighting. After 9 days, black pycnidia were present, which produced smooth, hyaline, linear to curved, filiform conidia, 4 to 6 septate (mostly 6), 31.8 to 70.1 × 2.0 to 2.8 μm. The morphological descriptions and measurements were similar to those reported for Dolabra nepheliae (3). The nucleotide sequence of the internal transcribed spacer (ITS) region including ITS1, 5.8S, and ITS2 intergenic spacers was determined for strain P11-1-1and a BLAST analysis of the sequence (GenBank Accession No. JX566449) revealed 99% similarity (586/587 bp) with the sequence of D. nepheliae strain BPI 882442 on N. lappaceum from Honduras. Based on morphology and ITS sequencing, the fungus associated with the cankers was identified as the same causal agent reported on rambutan and pulasan (N. mutabile) from Malaysia (1), and later reported on rambutan and litchi in Hawaii and Puerto Rico (3). Upon closer observations of the diseased samples, sections of corky bark contained at least two larval insects. The beetles were identified as Corticeus sp. (Coleoptera: Tenebrionidae) and Araecerus sp. (Coleoptera: Anthribidae) by the USDA-ARS Systematic Entomology Laboratory (Beltsville, MD). A corky bark disease on the trunk and larger limbs of mature langsat trees in Florida was thought to be caused by Cephalosporium sp. with larvae (Lepidoptera: Tineidae) feeding on the diseased tissue (2). It is not known the extent to which either of the beetle species is associated with L. domesticum in Hawaii or if they play a role in the bark disorder. To our knowledge, this is the first report of Dolabra nepheliae being found on langsat in Hawaii. Effective management practices should be established to avoid potential production losses or spreading the disease to alternative hosts. References: (1) C. Booth and W. P. Ting. Trans. Brit. Mycol. Soc. 47:235, 1964. (2) J. Morton. Langsat. In: Fruits of Warm Climates, p. 201-203. Julia F. Morton, Miami, FL, 1987. (3) A. Y. Rossman et al. Plant Dis. 91:1685, 2007.
In the last decade, rambutan (Nephelium lappaceum L., Sapindaceae) and pulasan (N. mutabile Blume) have been cultivated in Honduras to produce exotic fruits for export to North America (2). Recently, a disease was observed that produces dark brown to black fissured cankers from 1 to 3 cm long and 1 to 4 cm wide. The infected bark tissue becomes swollen with the middle region 3 to 8 mm thick. Symptoms appear when the trees are approximately 3 years old. As the trees mature, the cankers increase in size and weaken the branches, often resulting in breakage with the weight of the fruit causing substantial plant damage and fruit loss. In August 2010, fissured branch samples of rambutan and pulasan were collected from 6- to 8-year-old trees from the Humid Tropical Demonstrative Agroforestry Center in Honduras, Atlantida, La Masica (15°33'47.4″N, 87°05'2.5″W, elevation 106 m). A fungus associated with the cankers was identified as Dolabra nepheliae. It produces black, stipitate, elongate ascomata, 312 to 482 × 250 to 281 μm with broadly cylindric, bitunicate asci, 120 to 138 × 11.2 to 15.0 μm, and filiform, hyaline ascospores, 128 to 135 × 2.8 to 3.2 μm. Fungi from rambutan and pulasan were isolated on cornmeal agar plus 0.5% dextrose and antibiotics. On potato dextrose agar, the ascospores produced slow-growing colonies, 5 mm per week. In culture, isolates from both hosts produced pycnidia with elongated, slightly to strongly curved or S-shaped, hyaline conidia, 22.8 to 46.4 × 2.8 to 3.7 μm. This fungus was first reported on rambutan and pulasan from Malaysia (1,4), and later reported on rambutan and litchi in Hawaii and Puerto Rico (3). To our knowledge, this is the first report of D. nepheliae on pulasan and rambutan from Honduras. Specimens have been deposited at the U.S. National Fungus Collections (BPI 882442 on N. lappaceum and BPI 882443 on N. mutabile). Cultures were deposited at the Centraalbureau voor Schimmelcultures (CBS) as CBS 131490 on N. lappaceum and CBS 131491 on N. mutabile. Sequences of the internal transcribed spacer (ITS) region including ITS1, 5.8S, and ITS2 intergenic spacers were deposited in GenBank (Accession No. JQ004281 on N. lappaceum and Accession No. JQ004280 on N. mutabile). A BLAST search and pairwise comparison using the GenBank web server were used to compare ITS sequence data and recovered the following results: (i) CBS 131490 on N. lappaceum is 99% (538 of 544) identical to D. nepheliae CBS 123297 on Litchi chinensis from Puerto Rico; and (ii) CBS 131491 on N. mutabile is 99% (527 of 533) identical to the same strain of D. nepheliae. On the basis of the ITS sequence data, the isolates from Honduras were confirmed as the same species, D. nepheliae from Puerto Rico. Efforts to develop resistant germplasm and management strategies to control this disease have been initiated. References: (1) C. Booth and W. P. Ting. Trans. Brit. Mycol. Soc. 47:235, 1964. (2) T. Ramírez et al. Manual Para el Cultivo de Rambutan en Honduras. Fundación Hondureña de Investigación Agrícola. La Lima, Cortes, Honduras, 2003. (3) A. Y. Rossman et al. Plant Dis. 91:1685, 2007. (4) H. Zalasky et al. Can. J. Bot. 49:559, 1971.
A stem canker disease on rambutan (Nephelium lappaceum L.) and litchi (Litchi chinensis Sonn. (Sapindaceae) was found in plants in Hawaii and Puerto Rico. A fungus associated with cankers was identified as Dolabra nepheliae C. Booth & Ting (1). Numerous black, stipitate, elongate ascomata were produced within cracks of cankers. These ascomata contain elongate, bitunicate asci amid unbranched, interthecial elements and thin, cylindrical, hyaline ascospores measuring 96 to 136 × 2.5 to 3.5 μm. This fungus was originally described from Malaysia on N. lappaceum (1) and is also known on pulasan (N. mutabile Blume) in Australia (2). Classified by the Food and Agriculture Organization as a 'minor disease', the canker appears to be relatively common in Hawaii and was most likely introduced into Puerto Rico on imported germplasm. Nevertheless, efforts are underway to study the potential damage of this disease as well as mechanisms of control, including introduction of disease resistant clones. Specimens have been deposited at the U.S. National Fungus Collections (Hawaii on Nephelium BPI 878189, Puerto Rico (PR) on Nephelium BPI 878188, and PR on Litchi BPI 878190). Although a specimen of D. nepheliae on L. chinensis was collected from Hawaii in 1984 by G. Wong and C. Hodges and deposited as BPI 626373, this fungus was not known on Nephelium spp. in Hawaii and was not previously known from Puerto Rico on either host. References: (1) C. Booth and W. P. Ting. Trans. Brit. Mycol. Soc. 47:235, 1964. (2) T. K. Lim and Y. Diczbalis. Rambutan. Page 306 in: The New Rural Industries. Online publication. Rural Industries Research and Development Corporation, Australia, 1997.
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.
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.
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.