Identification of Plectosphaerella melonis from cucumber plants in Ukraine

Copyright: © 2021 E. Kopilov, H. Tsekhmіster, O. Nadkernychna, A. Kyslynska. This is an open access, peerreviewed article published by Firenze University Press (http://www.fupress. com/pm) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In Spain, Alfaro-García et al. (1996) reported that the causal agent of this disease, after the mass death of melons at the beginning of fruit ripening, was the fungus Acremonium cucurbitacearum. Bruton et al. (1996) described the same disease on melon plants in Texas (United States of America). Carlucci et al. (2012) determined that A. cucurbitacearum was synonymous to Nodulisporium melonis and transferred the pathogen to Plectosphaerella as P. melonis comb. nov.
A Plectosphaerella species was consistently isolated from diseased cucumber plants grown in a greenhouse in Ukraine. The aim of the present study was to identify the isolate fungus using morphological cultural and molecular genetic characteristics, and to confirm its pathogenicity to Cucumis sativus.

Plant sampling and fungus isolation
A fungus isolate (coded 502) was collected from affected roots of C. sativus (variety Koroliok), at the mass fruiting stage of crop growth. Cucumis sativus was grown in greenhouses in Chernihiv district, Chernihiv region (Ukraine). Affected host root segments (3-5 mm) were thoroughly washed for 15 min under running water, surface sterilized with 96% ethyl alcohol for 1 min then washed twice with sterile water, and were then plated into Petri dishes containing 4% barley meal agar (BMA) amended with 250 ppm streptomycin. The plates were incubated at 26°C, and after 4 d the resulting fungus was reisolated onto BMA.

Morphological identification
The cultural and morphological characteristics of the isolated fungus were examined after culture on BMA, Czapek's agar (CZA) or potato dextrose agar (PDA) in the darkness at 26°C. After 10 d in culture, the fungus completely covered the agar surfaces, and colonies were characterised.

Temperature/growth relationship
The temperature optimum for the isolated fungus was studied by culturing the fungus in Petri dishes on BMA at 10, 18, 26 or 35°C. After 10 d, the colony diameters were measured in two mutually perpendicular directions, and colony radial growth rates were determined (Pert, 1978).

Molecular identification
DNA extraction. For DNA isolation, parts of fungus colonies grown on BMA were processed using the AmpliSens DNA-sorb-B kit. The necessary quantity of DNA solution obtainment was carried out in appliance to described methods (Birnboim & Doly, 1979;Chowdhury & Akaike, 2005;Chi et al., 2009;Sika et al., 2015).
PCR analysis. For sequencing, obtained DNA solution polymerase chain reaction was carried out using ITS1 and ITS4 primers (White et al., 1990). The amplification reaction was conducted within Applied Biosystems equipment following prescribed methods (Watts & MacBeath, 2001;Garrido et al., 2009). Analysis of resulting 5.8S rDNA sequences were compared with GenBank database sequences using by BLAST analyses (http:// www.ncbi.nlm.nih.gov/blast).

Pathogenicity test
Pathogenicity tests were performed in a pot experiment using C. sativus plants (var. Nizhynskyi 12). Pots (2 L capacity) were each filled with 1800 g of soil which had been previously steamed at 70-80°C for 50 min.
Inoculum of the isolated fungus was prepared by growing the fungus on medium containing oat seeds, oat f lakes, water, chalk and gypsum in 1 L capacity f lasks. This substrate was sterilized twice at 128°C and 1.5 atm pressure for 1 h 30 min, and was then inoculated with pure cultures of the isolate, grown in tubes containing BMA. The f lasks were kept at 26°C for 21 d. Than the inoculum was transferred into paper bags, dried to constant weight at 30 °C, and mixed with soil.
Cucumis sativus seeds were sown to a depth of 2 cm. Fungus inoculum was applied to the pots at the equivalent of 5 × 10 4 CFU g -1 of soil (Bruton et al., 2000a). Experimental controls were cucumber plants grown without added inoculum. Soil in the pots was maintained at 60% of total water holding capacity. The pots were placed randomly in a greenhouse with natural lighting. The experiment was repeated five times. After emergence of the cucumber seedlings, these were thinned to five per pot. After 2 and 4 weeks, the plants were removed, washed from the soil and the root systems were evaluated.
Root segments (3-5 mm each) were thoroughly washed for 15 min under running water, surface sterilized with 96% ethyl alcohol for 1 min, and then washed twice with sterile water. The segments were then placed into Petri dishes containing 4% BMA plus 250 ppm Streptomycin at 26°C, and after 4 d the resulting fungus was reisolated onto BMA.

Statistical analyses
The data obtained were analyzed using Statistica 12. Normality of the data were assessed using Shapiro-Wilks W-test statistics, and homogeneity of variances was assessed using the Brown-Forsythe test. The nonparametric Mann-Whitney U test was applied (P ≤ 0.05) to compare experimental groups, including fungus growth media. One-way Analysis of Variance (ANOVA) and Duncan Multiple Range Tests were used to determine optimum temperature.
Isolate 502 was obtained from affected cucumber plants grown in greenhouses (Figure 1a). Disease symptoms were absent until the onset of the mass plant fruiting phase of cucumber. The symptoms included fading of leaf edges on the lower leaves of affected plants. The root systems had secondary and tertiary roots with necrotic areas (Figure 1b). No reductions in plant growth or delays in development were observed.
The macro-morphological features of isolate 502 differed depending on the culture medium ( Figure 2). Colonies were rounded, hyphae were thin, septate and hyaline. The colonies on PDA were white or cream, the colony surfaces were and velvety. On BMA, the colony colours varied from white to rose pink. On CZA the colonies had white aerial mycelium. Colonies had radial wrinkles on the reverse sides, and the colonies were slow-growing (Table 1), the most rapid growth was on PDA.
Phialides of isolate 502 were unbranched, septate, mostly simple, and measured from 22.4 × 4.2 μm to 42.0 × 4.2 μm. The phialides were coloured along their entire lengths. The phialides were located on hyphal filaments and each had a basal septum at the base. Unicellular conidia were assembled into a head at the apex of each phialide. Conidia were oblong, elliptical, smoothwalled and hyaline (granular). Conidia in PDA cultured measured 8.4 × 2.8 μm to 14.0 × 2.8 μm, and sporulation was most intense on this medium. On BMA, the conidia were smaller (5.6 × 3.1 μm to 11.2 × 2.8 μm) and sporulation was intense. On CZA, sporulation was not intense and the conidia were 4.2 × 4.2 μm to 8.4 × 4.2 μm. Chlamydospores were absent. These characteristics were similar to those described for P. melonis (Alfaro-Garcia et al., 1996;Carlucci et al., 2012).  The optimum temperature growth for isolate 502 was 26°C (Table 2).
Isolate 502 was deposited at the Depository of the Institute of Microbiology and Virology, NAS of Ukraine, with the number IMB F-100138.
Morphological identification of the isolate was confirmed by the molecular analysis. The sequence of isolate 502 was determined and submitted to GenBank with accession number MK736305.1. The BLASTn analysis showed 99% similarity with 14 strains of A. cucurbitacearum and P. melonis.
Pathogenicity of P. melonis 502 to cucumber plants was confirmed. Symptoms of root system damage were observed after 14 days for cucumber plants grown in soil inoculated with this isolate (Figure 3). The cucumber variety Nizhynskyi 12 was very sensitive to the pathogen at the two true leaf growth stage (after 2 weeks from sowing) (Figure 3c). Above-ground symptoms were absent after 14 d, although severe symptoms were apparent on the root system at this time. Investigating the sensitivity of cucurbit species to Spanish P. melonis isolates, Armengol et al. (1998) found that cucumber plants ranged from resistant to very susceptible, and that, in field conditions, cucumber plants were less affected by P. melonis than melon plants with the same level of susceptibility. In contrast, Bruton et al. (2000b), in studies of pathogenicity of American P. melonis isolates, concluded that cucumber plants belonged to a high-resistance host group.
The present results are the first to show pathogenicity of P. melonis on cucumber plants in Ukraine. This study has confirmed the pathogenicity of this fungus to cucumber, and has shown that young cucumber seedlings were very susceptible to this pathogen.