Identification and Pathogenicity of Globisporangium sp. Isolates Causing Tomato Stem Rot in Korea

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Res. Plant Dis. 2025;31(2):183-187

Publication date (electronic) : 2025 June 30

doi : https://doi.org/10.5423/RPD.2025.31.2.183

Gyo-Bin Lee¹, Sun Ha Kim², Sang-Keun Oh², Jae-Taek Ryu³, Wan-Gyu Kim^3,4,

¹Plant Disease Control Division, National Institute of Agricultural Sciences, Wanju 55365, Korea

²Department of Applied Biology, Chungnam National University, Daejeon 34134, Korea

³Bunong Seed Corporation, Suwon 16372, Korea

⁴Global Agro-Consulting Corporation, Suwon 16614, Korea

^*Corresponding author Tel: +82-31-292-7848 Fax: +82-31-292-7849 E-mail: wgkim5121@naver.com

Received 2025 June 2; Revised 2025 June 12; Accepted 2025 June 12.

Abstract

Tomato (Solanum lycopersicum) belongs to the family Solanaceae, and it is native to Peru (Plants of the World Online, 2025). The plant is grown worldwide as an important vegetable and food crop. In Korea, tomato is mostly cultivated in greenhouse conditions. The greenhouse cultivation of tomatoes forms favorable conditions for fungal disease development.

In March 2020, we observed unfamiliar stem rot symptoms in tomato plants grown for 40-50 days in trays in a greenhouse located in Pyeongchang, Korea. The symptoms initially developed on the basal stems and later extended up the stems above the soil line. The diseased part of the stems turned water-soaked, dark brown or black, and rotted (Fig. 1A, B). The diseased plants displayed wilt and died. Ten tomato trays in the greenhouse were observed, and 50 plants in each tray were investigated for stem rot outbreak. The in-vestigation revealed that the disease incidence on the plants ranged from 5% to 10%.

Fig. 1.

Stem rot symptoms in tomato plants. (A, B) Symptoms observed in the investigated greenhouse. Symptoms on varieties Dotaerang (C) and Seogwang (D) induced by inoculation tests with Globisporangium irregulare (SLPY-2003). Symptoms on varieties Dotaerang (E) and Seogwang (F) induced by inoculation tests with Globisporangium sp. (SLPY-2002). Non-inoculated plants Dotaerang (G) and Seogwang (H).

Diseased stems of tomato were collected from the investigated greenhouse, and fungi were isolated from lesions of the diseased stems. The 3-5 mm-long lesion pieces cut from the lesions were surface-sterilized with 1% sodium hypochlorite solution for 1 min. The lesion pieces were plated on 2% water agar, and hyphal tips growing from them were transferred to potato dextrose agar (PDA) slants after incu-bating the plates at 25^°C for 1 day. Four isolates presumed to be Pythium sp. were obtained from the lesion pieces and used for identification and pathogenicity tests.

The four isolates were cultured on V8-juice agar (V8A; V8 vegetable juice 200 ml, agar 20 g, distilled water 800 ml) in 9 cm diameter Petri dishes at 25°C in the dark for 5 days. Five mycelial disks (10 mm in diameter) were cut and removed from the V8A cultures, and then, 20 ml of sterile distilled water was poured into the culture dishes, which were incu-bated at 25°C in the dark for 5 days. The morphological characteristics of the isolates were examined by light microscope (Nikon Eclipse Ci-L, Tokyo, Japan) during the incubation.

The isolates produced non-septate hyphae, oogonia, antheridia, and oospores in the culture dishes (Fig. 2). Oogonia were globose, 15.5-22.0 μm (average 18.4 μm) in diameter, usually intercalary, smooth or with irregularly cylindrical and blunt projections. Antheridia were mostly monoclinous, occasionally diclinous, and 1-2 per oogonium. Antheridial cells were short, clavate, stalk long, and often falcate. Oospores were mostly aplerotic, globose, 15.2–20.4 μm (average 18.4 μm) in diameter, and with a wall 1.0-1.5 μm thick. The morphological characteristics of the isolates corresponded to those of the genus Globisporangium described in a previous study (Uzuhashi et al., 2010).

Fig. 2.

Morphological features of Globisporangium sp. isolates from tomato stems. (A, B) Antheridia, smooth oogonia and oospores. (C) An oogonium with irregular projections. (D) Antheridia, oogonia and oospores with irregular projections.

Phylogenetic analyses were conducted to identify species of the Globisporangium sp. isolates. Genomic DNA of the four isolates was extracted from mycelia cultured on PDA for 5-7 days using the CTAB method (Zhang et al., 2010). Amplification with polymerase chain reaction (PCR) for regions of internal transcribed spacer regions 1, 2 and intervening 5.8S nrDNA (ITS), cytochrome c oxidase subunit II gene (COX2), and b-tubulin gene (TUB) was performed using the DNA Free-Multiplex Master Mix (Cellsafe, Yongin, Korea) and primer sets of ITS1 and ITS4 for ITS (White et al., 1990), FM66 and FM 58 for COX2 (Martin and Tooley, 2003), and BT5 and BT6 for TUB (Villa et al., 2006). Amplification followed a protocol described in a previous study (Villa et al., 2006) and was purified using ExoSAP-IT™ (Applied Biosystems, Waltham, MA, USA). Subsequently, the PCR products were sequenced at Bionics Co., Ltd. (Seoul, Korea) with the same primers. The obtained sequences, along with sequences of other Globisporangium spp. (Salmaninezhad et al., 2022), were manually adjusted using SeqMan II (DNASTAR Inc., Madison, WI, USA) and aligned using MUSCLE (Edgar, 2004), with further refinement in MEGA version 7 software (Kumar et al., 2016).

Maximum likelihood analysis of the concatenated alignments of the three gene regions was conducted using a general time-reversible model performing 1,000 bootstrap replicates by MEGA version 7 software (Kumar et al., 2016). An outgroup taxon, Saprolegnia parasitica IFO 32780 was set. Among the four isolates of Globisporangium sp., one (SLPY-2003) clustered with a reference strain (CBS 493.86) of Globisporangium irregulare, and the others (SLPY-2001, SLPY-2002, and SLPY-2004) clustered with each other but not with any reference Globisporangium species (Fig. 3). It is suggested that the three isolates are likely to be a novel species. Accordingly, further detailed studies of the three isolates are needed to determine the species clearly. The sequence data of the isolates for ITS, COX2, and TUB were deposited in GenBank with the accession numbers OR259031-OR259034, PV601175-PV601178, and PV601179-PV601182, respectively.

Fig. 3.

Phylogenetic tree based on internal transcribed spacer regions 1, 2 and intervening 5.8S nrDNA, cytochrome c oxidase subunit II gene, and b-tubulin of Globisporangium sp. isolates (SLPY-2001, SLPY-2002, SLPY-2003, and SLPY-2004) from tomato stems and reference strains of Globisporangium spp. Sequence data of the reference species were obtained from the National Center of Biotechnology Information GenBank database. The phylogenetic tree was generated using the maximum likelihood method with a general time-reversible model. The bootstrap support values are shown at the nodes. The scale bar represents the number of nucleotide substitutions per site.

The isolates of G. irregulare and Globisporangium sp. were tested for their pathogenicity on tomato stems through artificial inoculation. For inoculation test, tomato seeds of two varieties (Dotaerang and Seogwang) were sown in trays in a greenhouse at 24-30^°C. The tomato seedlings grown in the trays were transplanted in circular plastic pots (height: 15 cm; upper diameter: 17 cm; lower diameter: 10 cm) 35 days after sowing. G. irregulare isolate (SLPY-2003) and Globisporangium sp. isolate (SLPY-2002) were cultured on millet medium (100 g millet : 100 ml distilled water) in 250 ml-flasks for 13 days to prepare the inoculum. The inoculum was inoculated to 47-day-old tomato plants of the two varieties that were grown in the circular plastic pots in a greenhouse. Surface soil around a tomato stem was dug at a depth of 2-3 cm, and 50 g of each inoculum was placed around the stem. The original soil was replaced on the inoculum, and the inoculated plants were cultivated in the greenhouse. The same quantity of millet medium without inoculum was used for the negative control plants. The inoculation test was repeated three times. Pathogenicity of the isolates was rated based on formation of stem rot symptoms on the inoculated plants 7 days after inoculation. The tested isolates of G. irregulare and Globisporangium sp. caused stem rot symptoms in the inoculated plants of the two varieties (Table 1, Fig. 1C-F), whereas no symptoms were found in the negative control plants (Fig. 1G, H). The stem rot symptoms induced by the artificial inoculation were similar to those observed in the investigated greenhouse. Morphologically identical strains were re-isolated from the stem lesions.

Table 1.

Result of pathogenicity tests of Globisporangium spp. isolates to tomato stems through artificial inoculation

Based on phylogeny and morphology, Uzuhashi et al. (2010) emended Pythium sensu stricto and described four new genera, Ovatisporangium, Globisporangium, Elongispo-rangium, and Pilasporangium. Uzuhashi et al. (2010) also revised Pythium irregulare to G. irregulare based on the phylogeny and morphology. Several Pythium spp. have been reported to cause damping-off or stem rot in tomato (French-Monar et al., 2014). In Korea, occurrence of damping-off in tomato caused by Pythium aphanidermatum was reported (Korean Society of Plant Pathology, 2025). However, there has been no report on occurrence of stem rot in tomato caused by Globisporangium spp. G. irregulare (synonym P. irregulare) has been reported to cause damping-off in melon (Park et al., 2020) and root rot in onion seedlings (Choi et al., 2023) in Korea. The present study is the first to reveal that G. irregulare and a new type of Globisporangium sp. cause tomato stem rot in Korea.

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