Wild rice (Oryza rufipogon Griff.) is the ancestor of AsianPublisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Castor (Ricinus communis L.) oil is used in the manufacture of cosmetics, lubricants, plastics, pharmaceuticals, and soaps and is grown in more than 40 countries with India and China leading in oil production(Tunaru et al. 2012). In June 2021, a seedling rot disease was observed on castor cv. Zibi-5 in a plant nursery in Zhanjiang (21°17’ N, 110°18’ E), China. Initial symptoms on leaves and stems were water-soaked and dark green lesions that resulted in rapid rotting. Disease incidence was 25% and resulted in seedling death. White fungal mycelia developed on the rotting plant tissues. Leaves and stems were collected from 10 diseased plants, surface disinfected in 0.5% sodium hypochlorite and 75% ethyl alcohol, and tissue pieces placed in plates of potato dextrose agar (PDA) which were maintained at 28℃. Hyphal tips from fungal mycelia that developed in the PDA plates were selected to establish pure cultures and three representative fungal isolates, designated RCC-1, RCC-2, and RCC-3, were selected for further study. The fungal isolates produced sporangiophores that were smooth, hyaline, aseptate, and apically swollen. Sporangiophores bore monosporous sporangiola that were broadly ellipsoidal, longitudinally coarsely striate, brown to dark brown, and measured 6.2 to 14.8 x 10.5 to 26.5 um (n=30). Sporangia contained few to many spores that were spherical, brown, and measured 59 to 150 um in diameter (n=20). Sporangiospores were ellipsoid, striate, and brown with multiple hyaline polar appendages and measured 6.6 to 12.3 x 10.6 to 25.5 um (n=30) in size. Based on these morphological characteristics, the fungus was identified as Choanephora cucurbitarum (Berk. & Ravenel) Thaxt. (Kirk, 1984). Molecular identification was done using the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) used to amplify the internal transcribed spacer (ITS) region and large subunit (LSU) with ITS1/ITS4 and NL1/LR3 (Walther et al. 2013). The amplicons were sequenced and the sequences were deposited in GenBank with accession numbers ITS, OL376748-OL376750, and LSU, OL763430-OL763432. BLAST analysis of these sequences revealed a 100% to 99% identity with the sequences (ITS, MG650194; 573/573, 573/573, and 573/573; LSU, AF157181; 673/676, 673/676, and 673/676) of C. cucurbitarum in GenBank. Pathogenicity tests, to fulfill Koch’s postulates, were performed in a greenhouse with a temperature range of 24℃ to 30℃ and 80% relative humidity. Thirty-day-old cv. Zibi-5 castor plants were grown in pots and used for inoculation tests. Ten plants were inoculated by placing agar plugs with mycelia of fungal isolate RCC-1 on leaves or stems. Ten control plants were inoculated with agar plugs only and the test was repeated three times in total. Five days after inoculation, all plants, with either leaf or stem inoculations, became infected and began rotting. Symptom progression was consistent with that observed in the nursery and all control plants remained healthy. C. cucurbitarum was successfully reisolated from all inoculated plants and identified by morphological characteristics and by sequence analysis. This fungus is known to cause serious damage on a wide range of hosts (Liu et al. 2019) and previously was reported on castor in India (Shaw 1984) and Papua New Guinea (Peregrin and Ahmad 1982). We observed that the pathogen grows very rapidly and causes serious damage to castor seedlings, warranting further investigation on the epidemiology and control of this disease.
Osmanthus fragrans Lin. is widely cultivated in China. Its flower is precious spices. It is also a garden ornamental plant. In March 2021, anthracnose-type lesions were observed on the leaves of O. fragrans in a public garden in Zhanjiang, Guangdong Province, China (21˚17'47''N, 110˚18'58''E). Disease incidence was around 50% (n = 100 investigated plants from about 30 hectares). The early symptoms were yellow spots on the edge or tip of the leaves. The spots gradually expanded and became dark brown, eventually coalescing into large irregular or circular lesions. Ten symptomatic leaves from 10 plants were sampled. The margins of the samples were cut into 2 mm × 2 mm pieces. The surfaces were disinfected with 75% ethanol for 30 sec and 2% sodium hypochlorite for 60 sec . Thereafter, the samples were rinsed thrice in sterile water, placed on PDA, and incubated at 28 ℃. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Thirty-two isolates of Colletotrichum ssp. were obtained (isolation frequency = 32/4×10 = 80%). Three representative single-spore isolates (OFC-1, OFC-2, and OFC-3) were used for further study. Colonies on PDA were white to gray with cottony mycelia in 6 days at 28 ℃. Conidia were one-celled, hyaline, cylindrical, clavate, and obtuse at both ends; they measured 10.5 to 17.5 µm × 3.5 to 5.0 µm (n = 50). Appressoria were oval to irregular in shape and dark brown in color, and they measured 6.5 to 8.5 µm × 4.5 to 7.5 µm (n = 20). Morphological characteristics matched the description of Colletotrichum siamense (Prihastuti et al. 2009; Sharma et al. 2013). For molecular identification, the colony PCR method (Lu et al., 2012) was used to amplify the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and actin (ACT) loci of the isolates using primer pairs ITS1/ITS4, GDF1/GDR1, CHS-79F/CHS-354R, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). Sequences of them deposited in GenBank under nos. MZ047368–MZ047370 (ITS), MZ126925–MZ126927 (GAPDH), MZ126895–MZ126897 (CHS-1), and MZ126835–MZ126837 (ACT). A phylogenetic tree was generated on the basis of the concatenated data from sequences of ITS, GAPDH, CHS-1, and ACT that clustered the isolates with C. siamense (the type strain MFLU 090230), while distanced the isolates with C. gloeosporioides (the type strain CBS 112999). The pathogenicity was tested through in vivo experiments. In group 1, the inoculation and control plants (n = 5, 3-month-old) were sprayed with a spore suspension (1 × 105 per mL) of the isolates and sterile distilled water, respectively, until run-off. In group 2, the unwounded leaflets were inoculated with mycelial plugs of the isolates or agar plugs (as control). Three plugs were for each leaflet ( n = 5). The plants were grown in pots in a greenhouse at 25°C to 28°C, with relative humidities approximately 80%. Anthracnose lesions were observed on the inoculated leaves after 10 days while the control plants remained healthy. The pathogen re-isolated from all the inoculated leaves was identical to the inoculation isolates in terms of morphology and just ITS analysis, but unsuccessful from the control plants. C. gloeosporioides has been reported to cause leaf spot on O. fragrans in Jiangxi Province of China (Tanget al., 2018), but not by C. siamense. To the best of our knowledge, this study is the first to report C. siamense causing anthracnose on O. fragrans. Thus, this work provides a foundation for controlling anthracnose in O. fragrans in the future.
Heteropanax fragrans (Roxb.) Seem is a common garden landscape tree in China. In December 2020, a leaf disease on H. fragrans was observed in a 2 ha field in Zhanjiang (20.85° N, 109.28° E), Guangdong province, China. Early symptoms were small yellow spots on leaves. Later, the spots gradually expanded and turned into necrotic tissues with a clear yellow halo and a white center. The disease incidence on plants was 100%. Twenty diseased leaves were collected from the field. The margin of the diseased tissues was cut into 2 mm × 2 mm pieces, surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, respectively, and rinsed thrice with sterile water before isolation. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. After 2-day incubation, grayish fungal colonies appeared on the PDA, then pure cultures were produced by transferring hyphal tips to new PDA plates. Single-spore isolation method was used to recover pure cultures for three isolates (HFA-1, HFA-2, and HFA-3). The colonies first produced a light-grayish aerial mycelia, which turned dark grayish upon maturity. Conidiophores were branched. Conidia numbered from two to four in chains, were dark brown, ovoid, or ellipsoid and mostly beakless; had 1–4 transverse and 0–3 longitudinal septa; measured within 7.2–17.8 (average = 10.2) × 2.5–7.5 (average = 4.3) µm (n = 30). Molecular identification was performed using the colony polymerase chain reaction method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) to amplify the large subunit (LSU), internal transcribed spacer (ITS) region, translation elongation factor (TEF) , and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with NL1/LR3, ITS1/ITS4, EF-1α-F/EF-1α-R, and GDF1/GDR1 (Walther et al. 2013;Woudenberg et al. 2015; Nishikawa and Nakashima. 2020). Amplicons of the isolates were sequenced and submitted to GenBank (LSU, ON088978-ON088980; ITS, MW629797, ON417005 and ON417006; TEF, MW654167, ON497264,and ON497265;GAPDH, MW654166, ON497262,and ON497263). The obtained sequences were 100% identical with those of Alternaria alternata strain CBS 102600 upon BLAST analysis . The sequences were also concatenated for phylogenetic analysis by maximum likelihood. The isolates clustered with A. alternata (CBS 102600, CBS 102598, CBS 118814, CBS 918.96,CBS 106.24, CBS 119543, CBS 916.96). The fungus associated with leaf yellow spot on H. fragrans was thus identified as A. alternata. Pathogenicity tests were conducted in a greenhouse at 24 ℃–30 ℃ with 80% relative humidity. Individual plants were grown in pots (n = 5, 1 month old). The unwounded leaflets were inoculated with 5 mm-diameter mycelial plugs of the isolates or agar plugs (as control). The test was performed thrice. Disease symptoms were found on the leaves after 7 days, whereas the controls remained healthy. The pathogen was re-isolated from infected leaves and phenotypically identical to the original isolates to fulfill Koch’s postulates. To our knowledge, this report is the first one on A. alternata causing leaf yellow spot on H. fragrans. Thus, this work provides an important reference for the control of this disease in the future.
Rhododendron pulchrum Sweet is a famous ornamental flower in China. In December 2020, a leaf spot disease was observed on cv. Maojuan in Zhanjiang (21.17 N, 110.18 E), Guangdong, China. The spots were irregular and distributed on both sides of the main vein. They were dark to black, and their borders were obvious. The coalescence of the spots eventually led to leaf wilt. The disease incidence was 100% (n = 100, about 50 ha ). Thirty infected leaves were collected from the field, and the margin of the diseased tissues was cut into 2 mm × 2 mm pieces. Samples were surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, respectively. They were rinsed thrice with sterile water before isolation. The tissues were plated on potato dextrose agar (PDA) medium and incubated at 28 ℃. After 5 days, fungal colonies appeared on the PDA. Pure cultures were produced by transferring hyphal tips to new PDA plates. Three isolates (RSP-1, RSP-2, and RSP-3) were obtained and the colonies of isolates were preserved in glycerol (15%) at -80 °C deposited at the Museum of Guangdong Ocean University. The morphology of these three isolates was consistent, and their sequences showed 100% homology according to ITS, TEF1, and ACT analysis results. The colonies grew to approximately 5 cm in diameter after 10 days. They showed olive green with off-white aerial mycelia. Stromata and conidia were observed on leaf lesions. Stromata were olivaceous brown. Conidia were solitary, cylindrical to narrowly obclavate, mildly curved, obtuse to rounded at the apex, and 1- to 3-septate; they had dimensions of 20 to 60 × 2.0 to 3.0 μm (n = 30). These morphological characteristics were not different from the description of Pseudocercospora rhododendricola (J.M. Yen) Deighton (Liu et al. 1998). For molecular identification, the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS), translation elongation factor 1-α gene (TEF1), and actin (ACT) loci of the isolates using primer pairs ITS4/ITS5, EF1/EF2, and ACT-512F/ACT-783R, respectively (White et al., 1990; O’Donnell et al. 1997). The sequences of the isolate RSP-1 were deposited in the GenBank (ITS, MW629798; TEF1, MW654168; and ACT, MW654170). BLAST analysis showed that the sequences of P. rhododendricola were submitted to GenBank for the first time by the author of this paper. A phylogenetic tree was generated based on the concatenated data of ITS, TEF1, and ACT sequences from GenBank by the Maximum Likelihood method. The isolates were closest to Pseudocercospora sp. CPC 14711 (Crous et al., 2013). Phylogenetic and morphological analyses identified the isolates as P. rhododendricola. Pathogenicity tests were conducted in a greenhouse at 24 °C–30 ℃ with 80% relative humidity. Healthy cv. Maojuan were grown in pots. Unwounded leaflets were inoculated with 5 mm-diameter mycelial plugs of the isolates or agar plugs (as control) (5 leaflets per plant, 3 plants, 2-month-old plants). The test was performed thrice. Disease symptoms were found on the leaves after 2 weeks, whereas the control plants remained healthy. The fungus was re-isolated from the infected leaves and confirmed as the same isolates by morphological and ITS analyses. P. rhododendricola was the cause of leaf spot of Rhododendron sp. from Singapore (Liu et al., 1998). For the first time, this pathogen was identified by combining phylogenetic and morphological analyses. The sequences in this study would be used as the reference sequences for further studies.
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