First report of Alternaria alternata causing leaf yellow spot on Heteropanax fragrans in China.

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 â„ƒ-30 â„ƒ 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.

First Report of Fusarium proliferatum Associated with Leaf Yellow on Alternanthera philoxeroides in China.

Alternanthera philoxeroides (Mart.) Griseb is a highly invasive weed commonly found in rice fields in China. In May 2021, leaf yellowing was observed on this weed (about 10 ha) in Zhanjiang (21°19'N, 110°20'E), Guangdong Province, China. Disease incidence was approximately 20% (n = 100 investigated plants). Ten yellow leaves from 10 plants were sampled, surface-sterilized with 75% ethanol for 30 s, followed by 2% NaClO for 5 min. The leaves were rinsed three times in sterile distilled water and four sections of each leaf were placed onto potato dextrose agar (PDA). Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty-two isolates of Fusarium ssp. (69% of the isolates) were obtained from 55% of the leaf samples. Three representative single-spore isolates (APF-1, APF-2, and APF-3) were used for further study. Colonies were white to pink on PDA. Conidiogenous cells were monophialidic or polyphialidic. Macroconidia were slightly curved, tapering apically with three to five septa, and measured from 32.5-55.8 µm × 2.5-5.1 µm in size (n=50). The morphological features of these fungi were noted to be in line with those of Fusarium proliferatum (Leslie and Summerell, 2006). For molecular identification, a colony PCR method (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS) and portions of elongation factor 1-α (EF1-α), RNA polymerase II largest subunit (RPB1), and RNA polymerase II second largest subunit (RPB2) genes using primers ITS1/ITS4, EF1-728F/EF1-986R, RPB1-R8/RPB1-F5, and RPB2-7CF/fRPB2-11aR, respectively (O'Donnell et al. 1998; O'Donnell et al. 2010). The sequences were submitted to GenBank under accession numbers MZ026797-MZ026799 (ITS) and MZ032209-MZ032217 (RPB1, RPB2, EF1-α). The sequences of the three isolates were 100% identical (ITS, 537/537 bp; RPB1, 1606/1606 bp; RPB2, 770/770 bp and EF1-α, 683/683 bp) with those of F. proliferatum (accession nos. MT378328, MN193921, MH582196, and MH582344) through BLAST analysis. Analysis of the sequences revealed a 99.87 - 100% identity with the isolates of the F. proliferatum (F. fujikuroi species complex, Asian clade) by polyphasic identification using the FUSARIUM-ID database (Yilmaz et al. 2021). The sequences were also concatenated for phylogenetic analysis by the maximum likelihood method. The isolates clustered with F. proliferatum. Pathogenicity was tested through in vivo experiments. The inoculated and control plants (n = 5, 30 days old) were sprayed with a spore suspension (1 × 105 per mL) of the three isolates individually and sterile distilled water, respectively, until run-off (Feng and Li. 2019). The test was performed three times. The plants were grown in pots in a greenhouse at 25 °C to 28 °C, with relative humidity of approximately 80%. Yellowing was observed on the inoculated plants after 7 days, while the control plants remained healthy. The pathogen re-isolated from all the inoculated plants was identical to the inoculated isolates in terms of morphology and ITS sequences. No fungi were isolated from the control plants. To the best of our knowledge, this study is the first to report F. proliferatum causing yellow symptoms on A. philoxeroides. The fungus has some potential biological control properties, but its host range needs to be further determined.

Pseudocercospora rhododendricola Causing Leaf dark Spot on Rhododendron pulchrum by the first phylogenetic analyses.

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 â„ƒ 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.