First Report of Collar and Root Rot of Lettuce Caused by Plectosphaerella cucumerina in Serbia

Abstract

In March 2021, unusual plant stunting and collar and wet root rot of lettuce (Lactuca sativa L.) during the rosette stage were observed in two commercial fields in Serbia (44°589N, 20°329E; 44°459N, 20°439E). Disease incidence in the fields ( 0.9 ha each) was approximately 15 and 20%, respectively. Initial above-ground symptoms were yellowing and wilting of leaves, while below-ground symptoms were collar and wet root rot and necrotic lesions. Eventually, whole plants wilted, collapsed, and died. A total of 35 symptomatic plants were collected from the fields and diseased tissues were cut into small pieces, surface sterilized, and plated on potato dextrose agar. Isolation resulted in 20 morphologically uniform monoconidial isolates. The isolates formed white to creamy colonies, which gradually became salmon pink, slimy, or moist in appearance, with sparse aerial mycelia. Numerous hyphal coils with conidiophores and hyaline, smooth-surfaced, ellipsoid to ovoid, septate or aseptate conidia were formed (4.5 to 10.1 × 1.2 to 3.7 μm [n = 100]). To confirm the species identity, the internal transcribed spacer (ITS) region and the D1/D2 region of a selected representative isolate 13-3-c were amplified and sequenced by using the primer pairs ITS1/ITS4 (White et al. 1990) and N1/N2 (O’Donnell and Gray 1995), respectively. The sequences were deposited in GenBank (ITS, OR880564; D1/D2, OR880567). Sequence analysis revealed 100% nucleotide identity with Plectosphaerella cucumerina isolates from different countries deposited in NCBI GenBank, including the isolates MH860704 (Vu et al. 2019) (ITS region) and KY662256 (Su et al. 2017) (D1/D2 region). Neighbor-joining analysis was conducted based on combined ITS and D1/ D2 regions, and the tree was constructed with the substitution models (1,000 bootstrap). Combined phylogeny confirmed that the sequences shared a common clade with P. cucumerina. Hence, morphological, microscopic, and molecular characterization confirmed the pathogen as P. cucumerina (Carlucci et al. 2012; Palm et al. 1995). In a pathogenicity assay, 10 isolates were tested. Five 30-day-old lettuce plants (cv. Majska Kraljica) per isolate were root-dipped in the conidial suspensions (1 × 105 conidia/ml). The 10 inoculated plants were transplanted in l-liter pots containing the sterile substrate (Floragard, Germany). Plants treated with sterile distilled water were used as controls. Plants were maintained in a greenhouse at 25 to 28°C under a 12-h photoperiod (Cai et al. 2021). Four weeks after inoculation, stunting, chlorosis, and wilting of plants were observed, while collars and roots exhibited typical decaying symptoms. No symptoms were observed on the control plants. The pathogen was reisolated from symptomatic tissue as previously described. Koch’s postulates were completed by confirming the identity of reisolates based on the morphological features. To our knowledge, this is the first report of P. cucumerina on lettuce or any other crops in Serbia. P. cucumerina is already known as a pathogen of lettuce and other hosts grown in many countries worldwide, as well as in some European countries (Belgium, England, Italy, Netherland, and Switzerland) (Zhang et al. 2019). This emerging pathogen may cause significant economic losses in lettuce production in Serbia and in the entire Balkan region. Our results may help to develop effective management strategies based on accurate and timely identification and regular pathogen monitoring.