First Report of Phyllosticta capitalensis Causing Black Spot Disease on Psidium guajava in Mainland China

Abstract

Guava (Psidium guajava) cv. Watermelon Bar was introduced into Guangxi province of China in 2015, and subsequently planted on more than 5,000 ha in Yulin city alone. Black spot disease on fruit was observed between June and October 2015. Once few diseased fruits were found on a guava tree, more than 30% fruits were found to be infected in a several days later. In the field, the fruits from more than 90% guava trees were infected by the disease. Spots containing pycnidia were dark brown to black, sunken, and 3 to 22 mm in diameter. Nine diseased guava fruits were collected from three trees in West Bank village (22.695387N, 110.112299E), Yuzhou District, Yulin City of Guangxi in China. Tissues from the margin between healthy and diseased portions of the fruit were excised and surface sterilized by immersing in 75% ethanol for 3 min, washed with sterile water three times, cut into 0.2 to 0.3 cm2 pieces, and placed on potato dextrose agar (PDA). After 3 days of incubation at 27℃, greyish-green fungal colonies developed. Hyphal tips were subcultured on PDA and OA (oat agar), incubated at 27℃ in a 12 h light/12 h dark cycle for 9 to 10 days until small black pycnidia appear under the mycelia, after which conidia were observed from pycnidia under the optical microscope. Pycnidia were black, granular and in clusters. Conidia were hyaline, unicellular, obovate, 7.47 to 14.49 μm × 5.01 to 9.63 μm, with mucoid sheath and apical mucilaginous appendage. After incubating for 20 days, black, granular and agglomerate perithecia under the mycelia were observed. Pressed the perithecia slightly on a slide, asci were squeezed out which were 8-spored, subcylindrical to clavate, 56.74 to 88.27 μm × 7.88 to 12.16 μm. Ascospores were hyaline, unicellular, multiguttulate, fusiform, wider in the middle, and 10.50 to 16.19 μm × 3.64 to 5.86 μm. Spermatia were hyaline, bacilliform with swollen ends, 3.15 to 8.99 μm × 1.02 to 1.88 μm. The single-spore isolate, YLWB01, was selected as one of 24 representative isolates, for molecular identification. DNA of the isolate YLWB01 was extracted, and the internal transcribed spacer (ITS) region, and the actin (ACT) and translation elongation factor 1 alpha (TEF) gene fragments were amplified using primers ITS1/ITS4 (White et al. 1990), ACT512F/ACT783R and ER728F/EF986R (Carbone et al. 1999), respectively. BLAST analysis in the GenBank database shown that obtained sequence of ITS (MN958712), ACT (MN958710) and TEF (MN958711) had 100% identify to Phyllosticta capitalensis (GenBank accession: AM403717, JN791558 and FJ538377, respectively). Phylogenetic analysis by neighbor-joining using MEGA 6.0 (Tamura. et al. 2013; Guarnaccia. et al. 2017) results shown that ITS, ACT and TEF sequences clustered together with P. capitalensis (strain CPC18848) in the phylogenetic tree. Based on these morphological characteristics and sequence analysis (Wikee et al. 2013), the fungus was identified as P. capitalensis, cause of guava black spot disease (Arafat 2018). Pathogenicity of YLWB01 single-spore isolate was demonstrated on fruits surface sterilized by 75% ethanol in the laboratory, six symptomless mature guava fruits were stab wounded, three stabs per fruit, then three fruits inoculated with a 5-mm-diameter colonized disk of PDA incubated for 10 days and three fruits inoculated with blank PDA disk as control. After inoculation, the fruits were incubated in glass covers at room temperature. Symptoms began to appear after 2 days and were similar to those of naturally infected guava fruits after 5 days. Control fruits remained healthy. The same fungus was re-isolated from the inoculated fruits, but not control fruits, using the methods described above. Guava black spot disease caused by P. capitalensis has been previously reported in Egypt (Arafat 2018). Zhang et al. (2011) also reported detection of P. capitalensis in China on imported guava fruit with black spots inspected by the Airport Office of Ningbo Entry-Exit Inspection and Quarantine Bureau in Zhejiang province of China, but not collected from the field. To our knowledge, this is the first report that P. capitalensis is the pathogen of guava black spot disease in mainland China.

Clonal Expansion in Multiple Phyllosticta Species Causing Citrus Black Spot or Similar Symptoms in China

“…Phyllosticta capitalensis is often reported as an endophyte or weak plant pathogen with a vast host range [15]. Recently, it was reported as the main pathogen of leaf spot on oil palm, Camellia sinensis, Ricinus communis, and fruit spot on Psidium guajava [49][50][51][52]. In this…”

“…Phyllosticta capitalensis is often reported as an endophyte or weak plant pathogen with a vast host range [15]. Recently, it was reported as the main pathogen of leaf spot on oil palm, Camellia sinensis, Ricinus communis, and fruit spot on Psidium guajava [49][50][51][52]. In this study, P. capitalensis strains were isolated from the leaves and fruits of various Citrus varieties with freckles or minute spots, and the pathogenicity test on fruits show slight symptoms, indicating that it might be a weak pathogen on citrus.…”

Morphological and Phylogenetic Analyses Reveal Three New Species of Phyllosticta (Botryosphaeriales, Phyllostictaceae) in China

“…The main distribution locations for these species are in America, Asia, and Europe, with the United States having the most extensive distribution [54,55]. Among the Phyllosticta species, P. carbitalensis, identified as a relatively weak plant pathogenic agent, is known to induce leaf spot diseases in various plants, including tea (Camellia sinensis), oil palm (Elaeis guineensis), Ricinus communis, and guava black spot [56,57] [8,14,33,44,45,[57][58][59][60] have notably enhanced the accuracy of Phyllosticta classification. This precision holds significant implications for the utilization and development of Phyllosticta species.…”

Morphological and phylogenetic analyses reveal two new species and a new record of Phyllosticta (Botryosphaeriales, Phyllostictaceae) from Hainan, China

“… Phyllosticta capitalensis is a cosmopolitan endophytic species reported in more than 300 host records in Fungal Databases ( https://nt.ars-grin.gov/fungaldatabases/index.cfm ) ( Okane et al 2001 , 2003 ; Baayen et al 2002 ; Glienke et al 2011 ; Wikee et al 2013b ; Wu et al 2014 ; Zhang et al 2015 ; Tran et al 2019 ; Hattori et al 2020 ). As a weak pathogen, P. capitalensis causes leaf spots on tea ( Camellia sinensis ), oil palm ( Elaeis guineensis ), Ricinus communis and black spot disease on Psidium guajava ( Cheng et al 2019 ; Nasehi et al 2019 ; Liao et al 2020 ; Tang et al 2020 ).…”