First report of Pythium root rot of hydroponic lettuce (Lactuca sativa) in Greece, caused by Pythium Cluster B2a sp

Abstract

Pythium root rot has been reported in several countries, but in Greece this disease was first detected in 2021, causing severe yield losses in a hydroponic lettuce crop. Isolations, morphological and molecular characterization, as well as pathogenicity assays identified a Pythium Cluster B2a species causing the disease in hydroponically grown lettuce. This is the first report of Pythium Cluster B2a sp. causing lettuce root rot in Greece.

Headnote

Summary. Pythium root rot has been reported in several countries, but in Greece this disease was first detected in 2021, causing severe yield losses in a hydroponic lettuce crop. Isolations, morphological and molecular characterization, as well as pathogenicity assays identified a Pythium Cluster B2a species causing the disease in hydroponically grown lettuce. This is the first report of Pythium Cluster B2a sp. causing lettuce root rot in Greece.

Keywords. Minor root pathogens, molecular characterization, pathogenicity assays.

INTRODUCTION

Hydroponic and soilless culture cropping systems occupy approx. 5% of the greenhouse area in Greece, with a tendency for further expansion, especially in new high-tech greenhouse facilities. The increasing interest in soilless production systems has led to development of specialized decision support systems, which provide full support in managing the nutrition of these crops through balanced nutrient solutions (Savvas et al., 2023). Lettuce and other leafy vegetables are mainly produced in open fields and high tunnel houses in Greece, but there is an increasing tendency for year-round production in modern greenhouses using hydroponic technologies such as the Nutrient Film Technique (NET) Barbosa et al., 2015). The lack of contact with soil in hydroponic lettuce production considerably reduces infections by soil-borne pathogens, although hydroponics cannot fully eliminate this risk.

Pythium root rot has been a major concern for hydroponic lettuce growers, and has been reported to cause important yield losses in Italy, Cyprus, and Connecticut, United States of America, especially when warm temperatures occur (Garibaldi et al., 2017; Pantelides et al., 2017; McGehee et al., 2018; Cacciola and Gullino, 2019). The disease has also been reported in hydroponi- cally grown Welsh onions in Japan (Shimizu and Tojo, 2021). Currently, there are no approved fungicides for the control of the disease, but research has been conducted on effects of some biological control agents and chemicals to control the disease (Utkhede et al., 2000).

MATERIALS AND METHODS

Sampling and isolation procedures

During September 2021, 30% of young 'Jokary' lettuce plants, grown hydroponically in an NET system located in Viotia region, Greece, exhibited dark brown necrotic lesions scattered throughout the root system at approx. 2 weeks post transplantation (Figure 1A). In mature plants, the whole root systems were necrotic, with reduced biomass compared to apparently healthy plants (Figure IB). In both cases, older leaves on the affected plants were chlorotic and wilted. Isolations were conducted onto V8-PAR medium, selective for oomycetes (Jeffers, 1986). For these, diseased roots were removed, washed with running tap water for 30 min and surface sterilized by immersion in 10% sodium hypochlorite for 2 min, 70% ethanol for 3 min, and then rinsing twice with sterile double distilled water. Small fragments of sterilized roots were placed onto V8-PAR medium and incubated at 25°C with a 12 h photoperiod.

DMA extraction and PCR amplification

Total genomic DNA was extracted from selected isolates according to Zelaya-Molina et al. (2011). An initial PCR assay was carried out using the universal primer set ITS5 and ITS4 (White et al., 1990) targeting the internal transcribed spacer region 1 and 2 containing the 5.8S region (Table 1). PCR products were precipitated using ammonium acetate and subjected to sequencing. The derived consensus sequences were edited using Benchling software (https://www.benchling.com/), and were compared to GenBank database sequences by BLASTn analysis. Due to the incapability of ITS region sequencing for identifying the isolated oomycete to the species level, additional molecular markers were employed. Cytochrome b oxidase subunits 1 and 2 (COU, COI2), which are accepted for oomycete barcoding, were amplified by PCR using appropriate primers (Table 1).

PCR products were purified and sequenced as described above. Sequences of ITS and cytochrome b oxidase subunits 1 and 2 were deposited in GenBank under the respective accession numbers OQ657948, OQ686764 and OQ686765.

Phylogenetic analysis

Phylogenetic analyses were conducted using the MEGA-X Software (Kumar et al., 2018). Phylogenies were inferred using the p-distance substitution model for the three loci using the Neighbor-Joining (NJ) statistical method and 1,000 bootstrap replications. Analyses were also run using the concatenated sequences of ITS, COU and COI2, utilizing the p-distance substitution model to construct phylogenetic trees using the NJ statistical method with 1,000 bootstrap replications.

Pathogenicity assays

Pathogenicity tests were conducted on 'Jokary' lettuce plants to investigate the role of the isolated oomycete species in disease development. Eight 2-week-old lettuce plants were grown in 60 x 39 x 20 cm (1/w/h) tanks (one with eight inoculated plants, and the other with eight non-inoculated plants), each containing a standard nutrient solution for commercial crops (pH = 5.6, EC = 2.63 dS m^-1, K+ 10.20 mmol L^-1, Ca2+ 4.86 mmol L1, Mg2+ 1.20 mmol L1, NH4+ 1.81 mmolL^-1, SO₄^2- 1.45 mmolL^-1, NO₃^- 16.78 mmolL^-1, H₂PO₄^- 1.36 mmolL^-1, Fe 62.27 mmolL^-1, Mn2+ 5 mmolL^-1, Zn2+ 4 mmolL^-1, Cu2+ 0.71 mmolL^-1, B 43.8 mmolL^-1, Mo 0.70 mmolL^-1, Cl^- 2.75 mmolL^-1, Na+ 0.20 mmolL1 and HCO₃^- 1.03 mmolL1). For oxygen flow and nutrient circulation, two air pumps were used in each tank to provide enough oxygen for the plants. For pathogen inoculum, two intact cultures of a Pythium Cluster B2a sp. isolate from 90 mm Petri dishes were mixed with 250 mL of nutrient solution and homogenized using a mixer. The mix containing the pathogen and nutrient solution was then poured into a tank (total volume of 3.5 L), while pure nutrient solution was added into a tank containing the non-inoculated control plants. The plants were then kept in a greenhouse at 25°C for 4 weeks.

RESULTS AND DISCUSSION

Forty-eight hours after isolation, oomycete-like colonies were observed on isolation plates, and these were transferred to new V8-PAR or PDA plates. The isolates produced white, flat, rosette-like mycelium on PDA (Figure 2), while on V8-PAR the mycelium was white and flat. Under microscopic observation, the colonies produced filamentous sporangia that formed dendroid structures and intercalary oogonia with straight oogonial stalks, of about 21 pm diameter. Antheridia were monoclinous and more than one antheridium per oogonium was observed. Oospores, while rarely developed, were plerotic or almost plerotic and uncoloured. Based on morphology, identification of the isolates to the species level was not possible, so molecular identification methods were used.

BLASTn analysis against GenBank database sequences revealed 100% identity of the isolates obtained with the Pythium species P. dissotocum, P. diclinum, P. coloratum and P. lutarium. According to Robideau et al. (2011), the above species belong to a group indicated as Pythium Cluster B2a, and these organisms are indistinguishable based only on their ITS regions. Phylogenetic analysis carried out with the three genetic loci separately or in concatenation produced similar topologies (Figure ЗА and 3B). Based on the evolutionary distances computed with the p-distance method for ITS locus and the concatenated sequences, the present study isolate belongs to the Pythium Cluster B2a species complex, and is closely related to P. diclinum with an evolutionary p-distance of 0 to 1,49 x 10^-3, respectively (Supplementary Table 1 and 2).

Approximately 10 days post-inoculation, plants inoculated with Pythium Cluster B2a sp. isolate showed chlorosis, yellowing and wilting symptoms of the lower leaves near the crowns, while their roots became brownish and eventually necrotic (Figure 4). Two weeks postinoculation, almost all the inoculated plants collapsed, while control plants showed no symptoms. To fulfill Koch's postulates, isolations were carried out from the inoculated and control plants into corn meal agar (CMA) containing tetracycline hydrochloride 0.01% v/v). Isolations from the inoculated plants yielded pure cultures identical to Pythium Cluster B2a sp. while no microbial growth was observed from the isolations conducted from the control plants, thus fulfilling Koch's postulates.

In some cases, Pythium species are considered as "minor root pathogens" (Stanghellini, 1986), but favourable conditions for the pathogen may lead to significant yield losses. Although prevalence of Pythium root rots in hydroponically grown lettuce is limited in Greece, it is important that the pathogen biology and the conditions leading to disease outbreaks are understood.

This is the first report of Pythium sp. belonging to Pythium Cluster B2a causing root rot in hydroponically grown lettuce in Greece.

Sidebar

Citation: C. Tsoukas, A. Venieraki, D. Savvas, E. Paplomatas(2023) First report of Pythium root rot of hydroponic lettuce (Lactuca sativa') in Greece, caused by Pythium Cluster B2a sp. Phytopathologia Mediterranea 62(3): 355-359. doi: 10.36253/phyto-14509

Accepted: September 7, 2023

Published: December 30, 2023

Copyright: ©2023C. Tsoukas, A. Venieraki, D. Savvas, E. Paplomatas. This is an open access, peer-reviewed 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.

Data Availability Statement: All relevant data are within the paper and its Supporting Information files.

Competing Interests: The Author(s) declare(s) no conflict of interest.

Editor: Thomas A. Evans, University of Delaware, Newark, DE, United States.

ORCID:

CT: 0009-0008-0628-9542

AV: 0000-0002-7954-7919

DS: 0000-0002-0064-7985

EP: 0000-0002-2929-0676

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