The wilt of winter cucumber in south-eastern Spain caused by Pythium irregulare

Pythium irregulare was isolated from adult cucumber plants with stem base necrosis, wilt or, frequently, that were dead; symptoms were particularly observed in crops grown in winter in plastic-houses in south eastern Spain. Damage was often important because symptoms develop during the fruit harvesting period. Cucumber plants cv. Nevada sown in late September were inoculated at the 4-7 leaf stage (mid October) with a suspension of propagules of P. irregulare isolated from diseased cucumber or tomato plants. Non-inoculated plants served as controls. Symptoms were recorded weekly until the end of the experiment in mid January and cumulative marketable yields calculated. The experiment was repeated the following year. A high incidence (93.1 and 97.9%) of diseased or dead plants occurred in plots inoculated with isolates of P. irregulare from cucumber, which were significantly more aggressive than tomato isolates. Plants in non-inoculated plots remained symptom free. Yields were significantly lower in plots inoculated with cucumber isolates in both years, mainly due to reductions in the number of fruits rather than fruit weight. This is the first report of P. irregulare as the causal agent of root rot in plastic-houses of cucumber plants in Almería (Spain), one of the world’s largest concentrations of plastic-houses. Additional key words: aetiology; Cucumis sativus; fruit yield; oomycetes; soil-less crops; zoosporic pathogens.


2009
).The substrates most commonly used in hydroponics are perlite bags and rockwool slabs.These crop systems were originally intended mainly to avoid root diseases (Zinnen, 1988;Stanghellini and Rasmussen, 1994a).However, elsewhere in the world and also here, soil-less systems also provide optimal conditions for zoosporic pathogens to spread and multiply rapidly (Stanghellini and Rasmussen, 1994a,b;Gómez, 2000) because Pythium spp.are well adapted to aqueous media (Hendrix and Campbell, 1973;Blancard et al., 1992;Martin and Loper, 1999).Long cucumber (Cucumis sativus) is widely cropped in south Mediterranean coastal provinces of Spain with 7,350 ha in 2008 (Anonymous, 2009), with ca.1,100 ha being cultivated in soil-less systems (García, personal communication).
Many Pythium spp.may cause damping-off of seedling in vegetables and may kill feeder roots but are seldom able to kill mature plants (Hendrix and Campbell, 1973).Cucumber seedlings are susceptible to Pythium aphanidermatum, P. ultimum, P. myriotylum, P. debaryanum, P. irregulare, P. sylvaticum, P. coloratum and isolates belonging to Pythium «group G».Symptoms usually comprise reduced growth, stem base and root necrosis and pre-emergence damping-off, with damage in artificial inoculation ranging from slight to complete plants loss, depending on the fungal species, the aggressiveness of isolates and environmental conditions (Jenkins and Averre, 1983;Favrin et al., 1988;Blancard et al., 1992;Moulin et al., 1994;Messiaen et al., 1995;Martin and Loper, 1999;Herrero et al., 2003).Specifically, P. irregulare caused damping-off at 20 and 24°C but not at 28 and 32°C (Ben-Yephet and Nelson, 1999).
Meanwhile, adult cucumber plants are susceptible to P. aphanidermatum, causing severe damage at temperature above 25°C, and P. ultimum is more aggressive at temperatures below 15°C, i.e. in non-heated greenhouses during winter.Others species, such as P. irregulare cause root necrosis and wilting during the hot hours of sunny days since the beginning of fruit harvesting until the end of the crop cycle, which appears to determine low yields (Favrin et al., 1988;Blancard et al., 1992).
P. aphanidermatum seems to be, for the commercial cucumber production, the most important pathogenic species of Pythium in different crop areas (Jenkins and Averre, 1983;Gold and Stanghellini, 1985;Favrin et al., 1988;Blancard et al., 1992;Messiaen et al., 1995), including the plastic-houses of the southern Mediterranean coast of Spain.In this area, dead cucumber plants occurring at any growth stage are frequently found, especially when they are grown in non-di-sinfested soil or in re-used substrates.P. aphanidermatum is prevalent during August-October and March-June for autumn-sown and spring-sown crops, respectively (Gómez, 2003).This work aimed to determine the aetiology of the disease that causes cucumber wilt and death during November to February in that area.
Two experiments were conducted in a plastic-house located at Campo de Dalías (36.76-36.81N, 2.58-2.92W), Almería, Spain; the plastic-house was covered with 200-µm thick transparent low density polyethylene film (Sotrafa, Almería, Spain).Pre-germinated seeds of cucumber cv.Nevada were sown directly in perlite bags in late September.Irrigation water had an electric conductivity of 0.5 to 0.6 dS m -1 ; a nutrient solution of 1.9 to 2.1 dS m -1 was prepared in a 25 m 3 tank for drip irrigation, with drainage of about 20% of the water volume used.Cucumber plants at the 4-7-leaf stage were inoculated with one of two mixtures each containing two isolates of P. irregulare, i.e., Py-14 plus Py-20 or Py-52 plus Py-53, obtained from diseased cucumber and tomato plants respectively.These isolates were identified on the basis of morphology of sporangia and oogonia (Van der Plaats-Niterink, 1981) and selected by the high mortality caused to cucumber seedlings and plants in winter (Gómez, 2003).For the two inoculation experiments conducted in greenhouse, the contents of two 90-mm diameter Petri dishes with potatocarrot-agar fully covered by colonies of the two isolates of each mixture were blended and homogenized with 500 mL distilled water to obtain concentrations of 1-5 10 2 colony-forming units mL -1 .Inocula were poured around cucumber plants at a rate of 50 mL plant -1 in mid-October.The experimental design was a complete randomised block with four replicates with individual plots comprising 18 plants (3 plants per perlite bag).Symptoms, mainly wilt, stem base necrosis and plant death were recorded weekly along the experimental periods.Weights and numbers of marketable fruits were recorded sequentially for the different plots from mid October to mid January and cumulative curves of cucumber fruit production calculated.Data of these variables for different inocula were analysed by ANOVA for each experiment (significance levels indicated as p-values in Table 1) and mean values were compared using Fisher's protected least significant difference (LSD) test at α < 0.05 significance level.
In experiment 1, the first symptoms of the disease, involving wilting of plants, were observed 45 days after inoculation with the mixture of P. irregulare isolates obtained from cucumber plants; plants inoculated with isolates from tomato developed symptoms a few days later (Fig. 1).Stem base necrosis was infrequent.Significantly higher (p < 0.05) incidences of diseased and dead plants were found when inoculation was with P. irregulare isolates from cucumber plants, whereas plants inoculated with those from tomato plants showed lower disease incidences.Non-inoculated plots remained disease free.
From day 26 to day 76 after planting the incidence of wilted plants rapidly increased to ca. 81% with P. irre-gulare isolates from cucumber and, simultaneously, to 68% in plants inoculated with isolates from tomato.Plant death first occurred when the maximum incidence of wilted plants was reached, and then rose sharply to 97% and 52% in plants inoculated with cucumber and tomato isolates respectively (Table 1).All inoculated plant showed symptoms of necrotic lesions on the roots.
Cumulative marketable yields showed significant (p < 0.05) reductions in the last 2-3 wk of the experiment in plants inoculated with P. irregulare isolates   from cucumber (Fig. 2).In contrast, cucumber yields did not differ between plots inoculated with P. irregulare isolates obtained from diseased tomato plants and non-inoculated (control) plots (Table 1).The final yield reduction for plants inoculated with P. irregulare isolates from cucumber plants averaged 16.3%, and was significantly greater (p < 0.05) in comparison with non-inoculated plants; this reduction was related to the number of fruits harvested and not to their weight (Table 1).In contrast, yield reductions in plots inoculated with P. irregulare isolated from tomato averaged only 4.1% and did not differ (p < 0.05) from the control plots (Fig. 2).
The development of the disease in experiment 2 followed a similar trend, although rates of wilted and dead plants were lower than those obtained in experiment 1 (Table 1; Fig. 1).The final marketable yield reduction for plants inoculated with P. irregulare isolates from cucumber plants averaged 36.7%, and was significantly greater (p < 0.05) in comparison with non-inoculated plants.Yield reductions in plots inoculated with P. irregulare isolated from tomato averaged 8.3% and did not differ (p < 0.05) from the control plots (Table 1).The pathogen was consistently recovered from symptomatic plants in both experiments and a significant reduction in the number of marketable fruits produced was observed when the plants were inoculated with the mixture of P. irregulare isolates recovered from diseased cucumber plants only, but fruit weight was not affected in any case (Table 1).
The pathogenicity of P. irregulare to adult cucumber plants, associated with the coldest months of the year, was demonstrated in both experiments by the frequent wilting, occasional stem base necrosis, and plant death.Symptoms did not appear until mid December when environmental conditions became favourable, but thereafter the epidemics developed rapidly.Also, yield reduction caused by P. irregulare during the months of the year when the fruit harvested reach a higher market value was demonstrated.The higher disease incidence observed in our experiments with the two cucumber isolates in comparison with tomato isolates may suggest a degree of isolate host specificity, but this requires verification with larger numbers of isolates from the two hosts.
The results differed from those of Moulin et al. (1994) who produced damping-off in cucumber seedlings inoculated with P. irregulare but failed to produce symptoms in adult cucumber plants; however they reported symptoms when adult cucumber plants were inoculated with P. aphanidermatum, probably because their incubation temperature was inadequate for P. irregulare infection.Disease caused by P. irregulare has been reported elsewhere e.g. in France, but only slight symptoms of root necrosis were reported (Blancard et al., 1992).However, our results are more in agreement with those obtained by Favrin et al. (1988), when cucumber plants were inoculated with P. irregulare and then incubated in a growth chamber, although the plants growth and environmental conditions in their experiments were completely different.
Under the winter conditions of SE Spain, the symptoms caused by P. irregulare on cucumber differ from those caused by P. aphanidermatum, with the latter species giving rise to severe damage at high temperatures (35°C) but causing only a moderate incidence of plant death and yield loss in autumn-sown crops (Gómez, 2003).In contrast, a high incidence of dead plants, but a lower incidence of stem base necrosis and relatively moderate yield production occurred with P. irregulare in winter (minimum and maximum temperatures ranging 8-10°C and 25-28°C, respectively).The low incidence of stem base necrosis observed in cucumber plants inoculated with P. irregulare seems to be due mainly to the age of the plants at inoculation rather than to the pathogen itself, and similar observations were made with P. aphanidermatum (Gómez, 2003).Future studies are needed to assess the distribution and control of the disease in winter cucumber crops grown in soil and in soil-less culture systems in plastic-houses of SE Spain.
Figure 1.Time-course of incidence of dead plants of cucumber cv.Nevada inoculated with a mixture of two isolates of Phytium irregulare obtained from diseased plants of cucumber (Cu) or tomato (To) (experiments 1 and 2).

Figure 2 .
Figure 2. Cumulative marketable yields of cucumber cv.Nevada inoculated with a mixture of two isolates of Phytium irregulare obtained from diseased plants of cucumber (Cu) or tomato (To) (experiments 1 and 2).

Table 1 .
Final incidence of diseased and dead plants and cumulative marketable yield of cucumber cv.Nevada inoculated with a mixture of two Pythium irregulare isolates from two host plants 1 In each column, values followed by the same letter are not statistically different (p < 0.05) according to Fisher's protected LSD test. 2 F values of the ANOVA.* Significant differences (p < 0.05).