Root rot of spinach caused by Pythium myriotylum and P. aphanidermatum in Taiwan.

Abstract

Spinach (Spinacia oleracea) is a commonly used green vegetable. During September and October in both 2022 and 2023, a vegetable nursery company located among paddy rice fields in Taichung City, Taiwan, reported significant failures in spinach seedling production in net-houses with mean outdoor temperatures of 28.7℃. Abnormal growth was observed in approximately 30% of the spinach seedlings in each batch (n = 2,000 to 3,000), with aboveground tissues showing stunting, yellowing, and wilt, and underground tissues displaying root rot. The symptoms resembled the spinach damping-off documented in Taiwan in extension articles but which lacked complete pathogen identification. A total of 110 plants from two batches were used for pathogen isolation by placing roots on water agar incubated at 25℃ or were examined for the presence of oospores in diseased roots. Eighty-one percent of these plants were associated with Pythium. Nine Pythium isolates were used in subsequent analyses. Genomic DNA from these isolates was subjected to amplification of ITS, β-tubulin gene (TUB2), and cytochrome C oxidase subunit Ⅱ (COXII) gene with primer pairs ITS1 / ITS4, BT5 / BT6, and FM58 / FM66 (Villa et al. 2006). Sequences of ITS (PP209187-PP209195), TUB2 (PP212864-PP212872), and COXII (PP212855-PP212863) were deposited in GenBank. Four isolates (sp01, sp02, sp03, and sp04) were 100% identical to the neotype strain (CBS 118.80) of Pythium aphanidermatum (Edson) Fitzp. for the ITS (761 bp), TUB2 (583 bp), and COXII (547 bp). Five isolates (2sp, 3sp, ND2-4sp, D3-4sp, and ND3-3sp) were 99.87%, 100%, and 99% identical to the reference strain (CBS 254.70) of Pythium myriotylum Drechsler for the ITS (762 bp), TUB2 (602 bp), and COXII (556 bp), respectively. Phylogenetic analysis of Pythium isolates inferred from concatenated sequences of the three genes (LéVesque and De Cock 2004; Villa et al. 2006) revealed that the same four isolates grouped with the neotype strain of P. aphanidermatum, and the five isolates clustered with the reference strain of P. myriotylum, each with a 100% bootstrap support. Morphological features of isolates ND3-3sp and sp01 were used for identification. Isolate ND3-3sp produced inflated lobulate sporangia and aplerotic and smooth oospores (16.3 to 25.1 um; n = 30) attached with three to five antheridia, consistent with identification as P. myriotylum. Isolate sp01 produced inflated lobulate sporangia and aplerotic and smooth oospores (17.0 to 24.0 um; n= 30) attached with a single intercalary antheridium, agreeing with the morphology of P. aphanidermatum (Van der Plaats-Niterink 1981). To investigate the pathogenicity of the nine Pythium isolates on spinach, 20 mycelial agar discs (4 mm in diameter) from a 2-day-old V8 culture of each isolate were used to induce sporangia and zoospores in 20 ml sterilized water at 25℃ with a 12 h light / dark regime. A 1.5 ml zoospore suspension (6 × 103 zoospores / ml) was dropped into BVB growth substrate of two spinach seedlings in 2-week-old at 25℃ with 12 h light / dark regime, resulting in symptoms resembling those observed in commercial nurseries at 7 days post-inoculation (dpi). Each Pythium isolate inoculated 20 seedlings in 10 cells of a planting tray. At 14 dpi, disease incidences were 95 to 100% for P. myriotylum isolates and 60 to 85% for P. aphanidermatum isolates, while control plants treated with water showed no symptoms. Re-isolated pathogens from the inoculated plants were morphologically identical to the inoculated isolates, completing Koch's postulates. Results of the pathogenicity assay, along with molecular and morphological identification, conclude that the root rot of spinach was caused by P. myriotylum and P. aphanidermatum. The two oomycetes were not formally documented to cause spinach diseases in Taiwan. Although P. myriotylum has been isolated from spinach (Wang et al. 2003), its pathogenicity to spinach was not documented worldwide. Root rot of spinach caused by P. aphanidermatum has been reported in the United States (Bates and Stanghellini 1984), Korea (Cho and Shin 2004), and Italy (Garibaldi et al. 2015). These pathogens thrive in humid and hot weather (Littrell and McCarter, 1970). Producing spinach in cooler weather or in a temperature-controlled environment may help prevent severe occurrence of the disease.